CN116615464A - Use of modulator of ITPRIPL1 in preparation of medicines for regulating immune response or anti-tumor - Google Patents

Abstract

Provided is the use of a modulator of ITPRIPL1 in preparing a drug for regulating immune response or anti-tumor, and the modulator is used to increase or decrease the expression or function of the ITPRIPL1 gene or protein of the body. Also provided is a pharmaceutical composition comprising the modulator employed in said use. Also disclosed are an isolated ITPRIPL1 recombinant protein, and an antibody that recognizes and binds ITPRIPL1. Based on the principle that ITPRIPL1 protein binds CD3ε and NRP2 receptors to regulate different immune cell functions, and then participates in the regulation of immune response and the immune escape process of tumors, it is confirmed that the modulator of ITPRIPL1 can be used to prepare medicines or pharmaceutical compositions, and can be used in tumor suppression, Conditions such as autoimmune diseases, transplant rejection, allergies and infections.

Description

Use of modulator of ITPRIPL1 in preparation of medicines for regulating immune response or anti-tumor

This application is required to be submitted to the China Patent Office on October 30, 2020, with the application number 202011191447.3, and the application name is "The use of modulators of ITPRIPL1 in the preparation of drugs for regulating immune responses or anti-tumor", and on May 24, 2021 The priority of the two Chinese patent applications filed with the Chinese Patent Office on 11th, the application number is 202110566040.2, and the application title is "isolated antigen ITPRIPL1 binding protein and its use", the contents of which are incorporated in this application by reference.

technical field

The application relates to the field of biomedicine, in particular to the use of ITPRIPL1 regulators in the preparation of drugs for regulating immune responses or antitumor.

Background technique

The protein encoded by ITPRIPL1 is Inositol 1,4,5-trisphosphate receptor-interacting protein-like 1 (inositol 1,4,5-trisphosphate receptor-interacting protein-like 1), and there has been no report on the function of ITPRIPL1 before. According to the annotation of UniProtKB database, human ITPRIPL1 includes 555 amino acids, which are divided into extracellular region (1-103 amino acids), transmembrane region (104-124 amino acids), and intracellular region (125-555 amino acids).

T cells are key effector cells of the adaptive immune response and have many important roles in pathogen elimination and autoimmune diseases. There are several subpopulations of T cells, each with a different function. The TCR (T cell receptor) found on the surface of T cells is a heterodimer composed of α and β polypeptide chains, constituting approximately 95% of the TCR population, or γ and δ polypeptide chains (Pitcher and van Oers, 2003 Year). Each polypeptide comprises constant (C) and variable (V) regions. The constant region is anchored in the cell membrane, while the variable region extends extracellularly and is responsible for binding antigens. The cytoplasmic short tail of the TCR lacks the ability to transduce signals. Intracellular signaling is initiated by the CD3 protein complex, which contains the intracellular immunoreceptor tyrosine activation motif (ITAM).

The CD3 (cluster of differentiation 3) T-cell co-receptor is a protein complex consisting of four distinct chains. In mammals, the complex contains one CD3 gamma (γ) chain, one CD3 delta (δ) chain and two CD3 ε (ε) chains. These chains are associated with TCR and delta chains (zeta chains), which generate activation signals in T lymphocytes. The TCR, delta chain and CD3 molecule together form the TCR complex. The CD3γ, CD3δ and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily comprising a single extracellular immunoglobulin domain. TCRs cannot bind free epitopes/antigens, instead, TCRs bind enzymatically cleaved fragments of larger polypeptides associated with the major histocompatibility complex (MHC), which is synonymous with the human human leukocyte antigen (HLA) system . This interaction occurs in a space known as the immune synapse. MHC class I molecules are expressed on all nucleated cells in the body and present antigens to cytotoxic T cells on which CD8 stabilizes the MHC/TCR interaction. Activation of cytotoxic T cells subsequently leads to destruction of target cells. MHC class II is found on macrophages, B cells and dendritic cells. These immune cells present antigens to helper T cells with CD4 that stabilizes MHC/TCR interactions. Interactions between MHC class II and TCRs ultimately lead to antibody-mediated immune responses. Other co-stimulatory molecules, such as CD45, CD28 and CD2, contribute to T cell activation at the immune synapse and initiate the formation of TCR signalosomes, macromolecular protein complexes responsible for intracellular signaling.

Several antibodies binding to human CD3ε have been reported, such as the antibody OKT3 (see for example Kung, P. et al., Science 206 (1979) 347-349; Salmeron, A. et al., J Immunol 147 (1991) 3047-3052) , the antibody UCHT1 (see eg Callard, RE et al., Clin Exp Immunol 43 (1981) 497-505) or the antibody SP34 (see eg Pessano, S. et al., EMBO J 4 (1985) 337-344). Of the currently known antibodies, SP34 is human cynomolgus cross-reactive (Conrad M.L. et al., Cytometry A 71 (2007) 925-933). Previously known proteins that directly bind to the extracellular region of CD3ε are antibody molecules, and there have been no reports of natural non-antibody proteins (such as typical ligands such as transmembrane proteins and secreted proteins) binding to the extracellular region of CD3ε.

NRP-2 (neuropilin-2) is a receptor that can regulate the function of immune cells (Am J Physiol Lung Cell Mol Physiol.2018), and it has been reported to regulate the function of antigen-presenting cells and promote tumor immune escape (Sohini Roy et al., Cancer Res.2018); NRP2 can affect immune cell migration, phagocytosis, and contact between immune cells (S Schellenburg et al., Mol Immunol.2017). The co-receptor formed by NRP2 and Plexin has a counter-chemotactic effect on the migration of lymphatic endothelial cells (Liu X et al., Cell Rep. 2016). NRP2 can also regulate the NFKB signaling pathway of cells, see (Rizzolio, S. et al. Cancer Research. 2017) and other reports. The previously discovered NRP2 ligands include members of the Semaphorin family, but other types of ligands have not been reported.

Contents of the invention

The purpose of this application is to provide a method for regulating immune response and suppressing tumors, and the use of modulators of ITPRIPL1 in the preparation of drugs for regulating immune response or anti-tumor.

In order to achieve the above purpose, the present application provides the use of a regulator of ITPRIPL1 in the preparation of drugs for regulating immune response or anti-tumor, the regulator is used to increase or decrease the expression or function of ITPRIPL1 gene or protein in the body.

Preferably, the regulator comprises any of the following:

(1) A gene editing system that knocks out or mutates the ITPRIPL1 gene in cells;

(2) an RNA molecule that reduces the expression level of the ITPRIPL1 gene;

(3) a nucleic acid molecule for introduction into cells, the nucleic acid molecule encodes ITPRIPL1 and increases the expression level of ITPRIPL1;

(4) isolated ITPRIPL1 recombinant protein;

(5) An antibody that recognizes and binds to ITPRIPL1.

Preferably, the gene editing system is a CRISPR/Cas9 gene editing system; the target sequence used for the CRISPR/Cas9 gene editing system is selected from any sequence shown in SEQ ID NO:11-13 for encoding sgRNA The oligomeric DNA sequence is selected from SEQ ID NO:14-19;

The nucleic acid molecule comprises: the sequence shown in SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10;

The ITPRIPL1 recombinant protein comprises: a functional fragment capable of binding to CD3ε or NRP2 protein in the extracellular region of ITPRIPL1 protein;

The antibody that recognizes and binds to ITPRIPL1 is a polyclonal antibody, a monoclonal antibody, a single chain antibody, an antigen binding domain, a bispecific antibody, a multispecific antibody, or an antigen binding part in a chimeric antigen receptor.

Preferably, the sequence of the functional fragment is selected from any one of SEQ ID NO: 1 to SEQ ID NO: 4, or its derivative sequence, the derivative sequence includes DRMDLDTLARSRQLEKRMSEEMRxLEMEFEERxxxAExxQKxENxWxGxTSxDQ ("x" is any amino acid), the The derivation methods include: replacing, deleting or inserting more than one amino acid without changing the function of the sequence.

Preferably, the ITPRIPL1 recombinant protein forms a fusion protein with an antibody constant region (as shown in SEQ ID NOs: 5-7) or forms a fusion protein with a coagulation factor; or, the ITPRIPL1 recombinant protein is modified, the modified Ways include: polyethylene glycol modification, glycosylation modification, polysialic acid modification, fatty acid modification, KLH modification, biotin modification.

Preferably, the nucleic acid molecule is introduced into cells through a drug delivery system, and the drug delivery system includes: recombinant expression vectors, viruses, liposomes or nanomaterials.

Preferably, the regulation of immune response includes: the regulation of the function of antigen presenting cells and T lymphocytes in the process of autoimmune response, suppression of transplant rejection immune response, allergic reaction, anti-infection immune response, anti-tumor immune response .

Preferably, the immune response includes: type I diabetes, immune infertility, rejection after organ transplantation, allergic reaction, systemic inflammation or cytokine storm, infection.

Preferably, the tumor is a solid tumor or a blood system tumor; the solid tumor includes: glioma, lung cancer, head and neck cancer, gastric cancer, colorectal cancer, thyroid cancer, esophageal cancer, urothelial cancer, Testicular cancer, breast cancer, cervical cancer, endometrial cancer, melanoma, pancreatic cancer or liver cancer; the blood system tumors include: leukemia or lymphoma.

The present application also provides a pharmaceutical composition, which comprises the modulator used in the above use, and a pharmaceutically acceptable carrier.

The present application also provides an isolated ITPRIPL1 recombinant protein, which is a functional fragment in the extracellular region of ITPRIPL1 protein that can bind to CD3ε or NRP2 protein.

The present application also provides an antibody that recognizes and binds to ITPRIPL1, and the antibody recognizes and binds to the extracellular region of the ITPRIPL1 protein.

The present application also provides the application of the isolated ITPRIPL1 recombinant protein, which is used to detect the existence of self-anti-ITPRIPL1 antibody, wherein the main steps of detection include directly contacting the ITPRIPL1 recombinant protein with the blood sample of the subject, and washing to remove non-specific binding.

The present application also provides the application of the above-mentioned antibody for detecting the content of ITPRIPL1 in a sample, and the antibody that recognizes and binds to ITPRIPL1 is used to judge the expression of ITPRIPL1 in a cell, tissue, organ or individual, or to judge whether it is suitable to administer the present application A method for modulating immune responses and suppressing tumors by targeting ITPRIPL1. In some embodiments, the antibody that specifically recognizes ITPRIPL1 can mark the boundary between cancer tissue and paracancerous tissue in the primary tumor, the boundary between cancer cells that have metastasized to lymph nodes and normal lymphoid tissue, and the distance between cancer cells that have metastasized and metastasis The boundaries of normal tissues of organs, and the application of labeling living cells of cancerous tissues in other biological samples.

The application provides an isolated antigen ITPRIPL1 binding protein used in the preparation of drugs for regulating immune response or anti-tumor and detecting the expression of ITPRIPL1 in individuals. The isolated antigen ITPRIPL1 binding protein can be combined with the antigen ITPRIPL1 as shown in SEQ ID NO :49 or the amino acid sequence shown in SEQ ID NO:1.

In certain embodiments, a heavy chain variable region and a light chain variable region are included, wherein the heavy chain variable region includes the amino acid sequence shown in any one of SEQ ID NO:24 or SEQ ID NO:34 HCDR1, HCDR2 and HCDR3 in the heavy chain variable region VH; said light chain variable region comprises amino acid sequence SEQ ID NO:25 or SEQ ID NO:35 in the light chain variable region VL shown in any one LCDR1, LCDR2, and LCDR3.

In certain embodiments, the amino acid sequence of the HCDR1 in the heavy chain variable region VH in the amino acid sequence SEQ ID NO:24 is as shown in SEQ ID NO:26, and the amino acid sequence of the HCDR2 is as shown in SEQ ID NO: 27, the amino acid sequence of the HCDR3 is shown in SEQ ID NO:28.

In certain embodiments, the amino acid sequence of the HCDR1 in the amino acid sequence SEQ ID NO:34 is shown in SEQ ID NO:36, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:37, and the amino acid sequence of the HCDR3 The amino acid sequence is shown in SEQ ID NO:38.

In certain embodiments, the amino acid sequence of the LCDR1 in the amino acid sequence SEQ ID NO:25 is shown in SEQ ID NO:29, the amino acid sequence of the LCDR2 is KV, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO : 31, or more than 80% similarity with the amino acid sequence shown in SEQ ID NO: 31.

In certain embodiments, the amino acid sequence of the LCDR1 in the amino acid sequence SEQ ID NO:35 is shown in SEQ ID NO:39, the amino acid sequence of the LCDR2 is KV, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO :41.

In certain embodiments, the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 in the heavy chain variable region VH shown in the amino acid sequence SEQ ID NO: 24; the light chain variable region includes the amino acid sequence SEQ ID NO: 24 LCDR1, LCDR2 and LCDR3 in the light chain variable region VL represented by ID NO:25.

In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region VH shown in the amino acid sequence SEQ ID NO:34; the light chain variable region comprises the amino acid sequence SEQ ID NO:34 LCDR1, LCDR2 and LCDR3 in the light chain variable region VL represented by ID NO:35.

In certain embodiments, it comprises an antibody heavy chain constant region, and said antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

In certain embodiments, it comprises an antibody light chain constant region, and said antibody light chain constant region comprises a human Ig kappa constant region.

In certain embodiments, it comprises an antibody heavy chain HC, and said HC comprises the amino acid sequence set forth in any one of SEQ ID NO:22 or 32.

In certain embodiments, it comprises an antibody light chain LC, and said LC comprises the amino acid sequence set forth in any one of SEQ ID NO:23 or 33.

In certain embodiments, it comprises an antibody or antigen-binding fragment thereof, wherein said antigen-binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv and/or di- scFv.

In certain embodiments, the ITPRIPL1 protein comprises human ITPRIPL1 or the ITPRIPL1 protein of a cynomolgus monkey, rat, mouse, gorilla, green monkey, golden snub-nosed monkey, black snub-nosed monkey, Amazon squirrel monkey.

In certain embodiments, the human ITPRIPL1 protein comprises the amino acid sequence shown in SEQ ID NO:20.

In another aspect, the present application also provides a chimeric antigen receptor (CAR), which comprises the isolated antigen ITPRIPL1 binding protein described in the present application.

In another aspect, the present application also provides an immunoconjugate comprising the isolated antigen ITPRIPL1 binding protein described in the present application.

In another aspect, the present application also provides one or more isolated nucleic acid molecules encoding the isolated antigen ITPRIPL1 binding protein or the chimeric antigen receptor described in the present application.

In another aspect, the present application also provides a vector comprising the nucleic acid molecule described in the present application.

On the other hand, the present application also provides a cell comprising the nucleic acid molecule or the vector described in the present application.

On the other hand, the present application also provides a pharmaceutical composition, which comprises the isolated antigen ITPRIPL1 binding protein described in the present application, the chimeric antigen receptor, the immunoconjugate, and optionally a pharmaceutical acceptable adjuvants.

On the other hand, the present application also provides a method for preparing the isolated antigen ITPRIPL1 binding protein described in the present application, the method comprising culturing the isolated antigen ITPRIPL1 binding protein described in the present application under the condition of expressing Apply to the cells as described.

On the other hand, the present application also provides the isolated antigen ITPRIPL1 binding protein described in the present application, the chimeric antigen receptor, the immunoconjugate, and/or the pharmaceutical composition described in the preparation Use in a medicament for preventing, alleviating and/or treating tumors. In certain embodiments, the tumors include solid tumors and lymphomas.

On the other hand, the present application also provides a linear epitope polypeptide that can be used for efficient screening and preparation of ITPRIPL1 function-modulating antibodies, characterized in that the peptide comprises: (i) the amino acid sequence is SEQ ID NO: 49, That is RLLEMEFEERKRAAE; (ii) or the amino acid sequence is or xxLxxxFxxRxxx (x is any amino acid), and 1-3 amino acids at both ends can be deleted; (iii) or 1, 2, 3, An amino acid sequence obtained by substitution, insertion or deletion of 4, 5, 6, 7, 8, 9 or 10 amino acids.

The above-mentioned linear epitope peptide has many outstanding advantages: it can be synthesized and prepared at a relatively low cost, and the function of the ITPRIPL1-specific antibody can be determined by analyzing the binding ability of the epitope peptide, which has the advantages of simple operation and stable and reliable detection results Advantages; linear epitope peptides can be directly injected into animals as immunogens or screened, so that more effective functional antibodies can be obtained than using full-length proteins, and the discovery efficiency of related drugs can be significantly improved.

The present application also provides a method for discovering and identifying ITPRIPL1 function modulators, which is characterized in that one or more of the following properties of the tested molecule are detected: the ability to specifically bind to ITPRIPL1 expressed on the cell surface; The effect on the binding of CD3ε; the effect on the combination of ITPRIPL1 and NRP2; the effect on the combination of ITPRIPL1 and SEMA3G; the effect on the combination of ITPRIPL1 and EBI2; the effect on the function of immune cells or tumor cells.

The applicant creatively developed a targeting antibody that can bind to ITPRIPL1, which can bind to the above-mentioned target protein with high affinity and neutralize its function, and inhibit its binding to one or more ligands, so as to relieve the immune escape function of tumor cells , to promote the killing of tumor cells by immune cells in vivo and in vitro; the antibody provided by the application can be used as an active ingredient to prepare a drug for treating tumors, providing a new effective solution for tumor treatment. At the same time, the invention also provides the linear epitope corresponding to the antibody with excellent neutralizing function and the biomarker of the administered antibody.

The idea, specific structure and technical effects of the present application will be further described below in conjunction with the accompanying drawings, so as to fully understand the purpose, features and effects of the present application.

Compared with the prior art, the present application has the following beneficial effects:

The present application discloses a method for regulating immune response and suppressing tumor, and the above purpose is realized by regulating the expression or function of ITPRIPL1 gene. This application is based on the new scientific discovery that ITPRIPL1 binds to CD3ε and other proteins to regulate the functions of different immune cells, and then participates in the regulation of immune responses and the immune escape process of tumors. The present application proves that the modulator of ITPRIPL1 can be used to prepare medicine or pharmaceutical composition, and has application prospects in suppressing diseases such as tumor, autoimmune disease, transplant rejection, allergy and infection.

Description of drawings

Fig. 1 is the plasmid construction result of embodiment 1;

Fig. 2 is the co-immunoprecipitation experimental result of embodiment 1;

Fig. 3 is the result diagram of co-localization of exogenously expressed ITPRIPL1 and CD3E in the cells of Example 1;

Fig. 4 is the construction result of the expression vector expressing the ITPRIPL1 extracellular region receptor binding domain of Example 2;

Fig. 5 is the co-immunoprecipitation experimental result of embodiment 2;

Figure 6 shows the experimental results of immunofluorescence and colocalization analysis of ITPRIPL1 extracellular region and CD3 extracellular region in trans binding in Example 2;

Figure 7 is the result of Coomassie Brilliant Blue staining of the ITPRIPL1-RBD recombinant protein (IT1-RBD protein) gel electrophoresis in Example 3 (Note: ITPRIPL1=IT1, the same below);

Fig. 8 is the ELISA experimental result of embodiment 4;

Figure 9 and Figure 10 are the results of flow cytometry in Example 5 demonstrating that the purified protein fragment of the extracellular region of ITPRIPL1 binds to Jurkat cells that highly express CD3, wherein (a) in Figure 9 is the threshold setting for not dividing into dead cells Determined, (b) of Figure 9 reflects the binding situation of Jurkat cells and different concentrations of purified protein fragments of the extracellular region of ITPRIPL1. Figure 10 is the specific staining and protein binding conditions under each condition in (b) of Figure 9;

Figure 11 and Figure 12 are the results of flow cytometry in Example 5 demonstrating that the ITPRIPL1 protein binds to cells overexpressing CD3 with higher efficiency, wherein (a) in Figure 11 is the threshold setting for not dividing into dead cells, (b) of FIG. 11 reflects the combination of cells with different CD3ε expression and different concentrations of ITPRIPL1 recombinant protein. Figure 12 is the specific staining and protein binding conditions under each condition in (b) of Figure 11;

Figure 13 and Figure 14 are the results of flow cytometry in Example 5 demonstrating that CD3 binds to cells overexpressing ITPRIPL1 with higher efficiency, wherein (a) in Figure 13 is the threshold setting for not dividing into dead cells, Figure 13 (b) reflects the combination of cells with different ITPRIPL1 expression and CD3ε protein. Figure 14 is respectively the specific staining and protein binding conditions under each condition in (b) of Figure 13;

Figure 15 is a graph showing the results of NFKB signal changes with ITPRIPL1 protein concentration under the condition of 50 micrograms per milliliter of ConA activation in Example 5;

Fig. 16 is the result figure of the NFKB signal change with the microsphere-coated ITPRIPL1 protein concentration change under the condition of 50 micrograms per milliliter of ConA activation in embodiment 5;

Figure 17 shows the expression of ITPRIPL1 in different types of tumor cells after the internal reference of GAPDH is adjusted in Western blotting;

Figure 18 shows the mRNA expression level of ITPRIPL1 in normal tissue and tumor tissue;

Fig. 19 is the result figure that the enzyme-linked immunosorbent assay in embodiment 6 proves that the polyclonal antibody can bind to cells expressing ITPRIPL1;

Fig. 20 is the result figure that the enzyme-linked immunosorbent assay in embodiment 6 proves that the polyclonal antibody can block the combination of ITPRIPL1 and CD3E;

Figure 21 and Figure 22 are the results of flow cytometry in Example 6 demonstrating that polyclonal antibodies can block the binding of ITPRIPL1 to cells overexpressing CD3ε, wherein (a) in Figure 21 is the threshold setting for not dividing into dead cells Certainly. (b) of Figure 21 reflects the changes in the binding between ITPRIPL1 and CD3ε when the concentration of the polyclonal antibody changes. Figure 22 is the specific staining and protein binding conditions under each condition in (b) of Figure 21;

Fig. 23 is a result diagram showing that the luciferin reporter experiment in Example 7 proves that HCT116 cells overexpressing ITPRIPL1 can more reduce the NFKB proliferation signal of Jurkat-dual cells;

Figure 24 is a result diagram showing that the luciferin reporter experiment in Example 7 proves that the CD3E protein can block the inhibition of the ITPRIPL1 protein on the NFKB proliferation signal of Jurkat-dual cells;

Figure 25 and Figure 26 are the results of flow cytometry in Example 8 proving that the ITPRIPL1-RBD recombinant protein can reduce the killing of human peripheral blood mononuclear cells (PBMC) to kidney-derived H3K293 cells, wherein (a) of Figure 25 and (b) indicate the division of 293E cells according to CD45. (c) of FIG. 25 is the relative killing activity of PBMC calculated based on the apoptosis data of each group under different ITPRIPL1 protein concentration conditions. Figure 26 is the specific apoptosis staining situation under each condition in (c) of Figure 25;

Figure 27 and Figure 28 are flow cytometry in Example 9 demonstrating that ITPRIPL1 overexpression can reduce the killing of PBMCs to tumor cells and knocking out ITPRIPL1 can promote the results of PBMCs' killing of tumor cells, wherein (a) and Figure 27 (b) represents the division of HCT116 cells according to CD45. (c) of FIG. 27 is the relative killing activity of PBMC calculated based on the apoptosis data of each group under different polyclonal antibody concentration conditions. Figure 28 is the specific apoptosis staining situation under each condition in (c) of Figure 27;

Figure 29 and Figure 30 are the results of flow cytometry in Example 10 demonstrating that the ITPRIPL1 polyclonal antibody can promote the killing of tumor cells by PBMC. (a) and (b) of FIG. 29 show that HCT116 cells were divided according to CD45. (c) of FIG. 29 is the relative killing activity of PBMC calculated based on the apoptosis data of each group under different polyclonal antibody concentration conditions. Figure 30 is the specific apoptosis staining situation under each condition in (c) of Figure 29;

Figure 31 is the co-immunoprecipitation experiment result of embodiment 11;

Figure 32 is a graph showing the results of the enzyme-linked immunosorbent assay in Example 12;

Figure 33 and Figure 34 are the experimental results of flow cytometry in Example 12 demonstrating that NRP2 binds to cells overexpressing ITPRIPL1 with higher efficiency. Among them, (a) of FIG. 33 is the threshold setting for not classifying dead cells. (b) of Figure 33 reflects the binding situation of cells with different expression of ITPRIPL1 and NRP2 protein, and Figure 34 shows the specific staining and protein binding situation under each condition in (b) of Figure 33;

Figure 35 is the result of the luciferin reporter experiment in Example 13;

Figure 36 is the result of the luciferin reporter experiment in Example 14;

Fig. 37 and Fig. 38 are the results of flow cytometry in Example 14 demonstrating that the purified IT1-RBD1-Fc recombinant protein can reduce the killing of human peripheral blood mononuclear cells (PBMC) to kidney-derived H3K293 cells, wherein , (a) of FIG. 37 shows that 293E cells were divided according to CD45. (b) of Figure 37 is the relative killing activity of PBMC calculated based on the apoptosis data of each group under different ITPRIPL1 expression and different protein conditions, and Figure 38 is the specific apoptosis staining situation under each condition in (b) of Figure 37;

Fig. 39 is the result of the immunoblotting experiment of embodiment 14;

Figure 40 shows that the OCTET molecular interaction in Example 5 proves that the ITPRIPL1 protein can directly bind to the CD3E protein;

Figure 41 is the result of immunoblotting experiment of CD3ε protein blocking the effect of ITPRIPL1-RBD-Fc recombinant protein on the phosphorylation pathway in Example 14;

Figure 42 is the Western blot experiment result of the influence of the CD3 mutant of Jurkat on the phosphorylation pathway in Example 15;

Figure 43 is the result of the immunofluorescence experiment of the ITPRIPL1-RBD-Fc recombinant protein on the calcium ion flow in Jurkat cells in Example 15;

Fig. 44 is the immunofluorescence experiment result that the CD3 mutant of Jurkat in embodiment 15 affects different responses to intracellular calcium ions of ITPRIPL1-RBD-Fc recombinant protein;

Figure 45 is the result of immunoblotting experiment in which the ITPRIPL1-RBD-Fc recombinant protein increases CD3 binding to Nck in Example 16;

Figure 46 is the result of the proximity junction experiment in which the ITPRIPL1-RBD-Fc recombinant protein increases the binding of CD3 and Nck in Example 16;

Figure 47 is the experimental results of tumor volume monitoring and tumor weight measurement of the humanized CD3ε mouse MC38 subcutaneous xenograft tumor model in Example 17;

Fig. 48 is the result of flow cytometry experiment of the humanized CD3ε mouse MC38 subcutaneous xenograft tumor model in Example 17, which was sacrificed and collected from PBMCs for analysis of T cell-related immune regulation points;

Figure 49 is the immunohistochemical results of the tumor tissue of the humanized CD3ε mouse MC38 subcutaneous xenograft tumor model in Example 17;

Figure 50 is the flow cytometry experiment result of the analysis of T cell-related immune regulatory points in PBMCs of ITPRIPL1 knockout and wild-type mice in Example 17;

Figure 51 is the results of ELISA analysis of secreted cytokines in PBMCs of ITPRIPL1 knockout and wild-type mice in Example 17;

Figure 52 shows the results of immunohistochemical staining of T cells in testicular tissue of ITPRIPL1 knockout and wild-type mice in Example 17, and the results of sperm morphology and motility analysis;

Figure 53 is the analysis result of the expression of ITPRIPL1 in tumor and normal tissue in Example 18;

Figure 54 shows the ITPRIPL1 sequence and functional annotations described in Example 14, the bar graph is the combination of different species;

Figure 55 is the labeling and distinguishing effect of ITPRIPL1 antibody on tumor tissue and normal tissue;

Figure 56 is the specific labeling and distinguishing effect of ITPRIPL1 antibody on tumor cells with distant metastasis;

Figure 57 shows the results of the combination of mouse hybridoma antibodies and ITPRIPL1 in the present application, wherein Figure 57A is an ELISA result of the reaction of 1 microgram per milliliter of 100 hybridoma antibodies with 1 microgram per milliliter of ITPRIPL1, and Figure 57B is 1 The ELISA result graph of the reaction of 9 micrograms per milliliter of selected hybridoma antibodies with 1 microgram per milliliter of ITPRIPL1. Figure 57C is a graph showing the binding curve of 13B7 antibody to ITPRIPL1;

Figure 58 shows the results of detecting the binding of each mouse hybridoma antibody to endogenous Jurkat cells with high expression of ITPRIPL1 by flow cytometry analysis technology in the present application, wherein Figure 58A is the Jurkat cell circle gate setting, and Figure 58B is each Statistical results of hybridoma antibody binding rate. Figures 58C-58L show the results of flow cytometric analysis of the binding of different hybridoma antibodies 2E7, 5E5, 13B7, 13F7, 15C9, 16E1, 18B12, 18G5, 19B11 and 20E3 to Jurkat cells;

Figure 59 shows the results of the detection of mouse hybridoma 13B7 antibody binding to various tumor cells expressing ITPRIPL1 by flow cytometry in this application. Figure 59A is the control without adding antibodies to HCT116, and Figure 59B is the group of adding antibodies to HCT116. Figure 59C is A549 without adding antibody control, Figure 59D is A549 adding antibody group, Figure 59E is MC38 without adding antibody control, Figure 59F is MC38 adding antibody group, Figure 59G is MC38-ITPRIPL1 stable transfection without adding antibody control, Figure 59H The antibody group was added to the MC38-ITPRIPL1 stable transfection. Figure 59I is the Jurkat without antibody control, Figure 59J is Jurkat with the antibody group, Figure 59K is Raji without the antibody control, Figure 59L is Raji with the antibody group, and Figure 59M is the 13B7 antibody Statistical graph of the binding rate to different tumor cells expressing ITPRIPL1;

Figure 60 shows the results of the binding of different concentrations of mouse hybridoma 13B7 antibody to endogenous Jurkat cells highly expressing ITPRIPL1 detected by flow cytometry in the present application, wherein Figure 60A is the gate setting and Figure 60B is negative For comparison, Figure 60C-Figure 60H are the binding rates of 0.0625/0.125/0.25/0.5/1/2 micrograms per milliliter of 13B7 antibody binding respectively, and Figure 60I is the statistical results of the binding rates of each group;

Figure 61 shows the results of Western blot analysis of the binding of 13B7 antibody to ITPRIPL1 in this application, in which Jurkat cells endogenously expressing ITPRIPL1, HCT116 cells endogenously expressing ITPRIPL1 and MC38 cells not expressing ITPRIPL1 were used Perform Western Blot experiments and incubate with 13B7 antibody;

Figure 62 shows the results of different mouse hybridoma antibodies blocking the binding of ITPRIPL1 to different proteins in the present application, wherein, Figure 62A shows the results of blocking the binding of ITPRIPL1 to CD3E by different mouse hybridoma antibodies, and Figure 62B shows the different The results of blocking the binding of ITPRIPL1 and SEMA3G by the mouse hybridoma antibody;

Figure 63 shows the ELISA results of the mouse hybridoma monoclonal antibody binding to ITPRIPL1 in the present application;

Figure 64 shows the results of the flow cytometry analysis technology in this application detecting the binding of different mouse hybridoma monoclonal antibodies to endogenous Jurkat cells highly expressing ITPRIPL1;

Figure 65 shows the statistical results of different mouse hybridoma monoclonal antibodies blocking the binding of ITPRIPL1 to different proteins and the sequence comparison of the two antibodies, wherein Figure 65A shows different mouse hybridoma monoclonal antibodies blocking The results of the combination of ITPRIPL1 and CD3E, Figure 65B shows the statistics of different mouse hybridoma antibodies blocking the combination of ITPRIPL1 and SEMA3G, Figure 65C shows the comparative analysis of the two antibody sequences;

Figure 66 shows the results of identification of the antigen-binding region of ITPRIPL1 in this application. Among them, Figures 66A-66M are sequentially the statistical results of the binding of antibodies 18B12, 18B12D1A6, 13B7, 13B7A6H3, 16E1, 18G5, 20E3, 16E1D8H1, 5E5, 2E7, 19B7, 13F7, 18G5F3F4 to different peptides from ITPRIPL1 protein;

Figure 67 shows the cell flow analysis detection results of different mouse hybridoma monoclonal antibodies in the present application to promote the killing of Raji cells endogenously expressing ITPRIPL1 by PBMC, wherein, Figure 67A-B is the circle gate setting, Figure 67 67C is the Raji cell self-apoptosis control, Figure 67D is the killing situation of adding PBMC and negative serum, Figure 67E-Figure 67N are respectively adding 0.5 micrograms per milliliter of 13B7A6H3 monoclonal antibody and 2 micrograms per milliliter of 13B7A6H3 monoclonal antibody when adding PBMC Antibody, 0.5 μg/ml 16E1D8C4 mAb, 2 μg/ml 16E1D8C4 mAb, 0.5 μg/ml 18G5F3E5 mAb, 2 μg/ml 18G5F3E5 mAb, 0.5 μg/ml 18B12D1 mAb Antibody, 2 micrograms per milliliter of 18B12D1 monoclonal antibody, 0.5 micrograms per milliliter of 18B12D1A6 monoclonal antibody, and 2 micrograms per milliliter of 18B12D1A6 monoclonal antibody;

Figure 68 shows the statistical chart of the cell flow analysis detection results of different mouse hybridoma monoclonal antibodies in the present application promoting the killing of Raji cells endogenously expressing ITPRIPL1 by PBMC;

Figure 69 shows the results of the ELISA experiment on the binding of the P8 polypeptide segment to the 13B7A6H3 monoclonal antibody after undergoing different point mutations in this application;

Figure 70 shows the experimental results and corresponding antibody grouping analysis using the epitope mapping method in this application;

Figure 71 shows the experimental results of the tumor volume and quality changes in the subcutaneous xenograft tumor model overexpressing MC38-ITPRIPL1 in mice treated with a monoclonal antibody combined with ITPRIPL1-RBD;

Figure 72 shows the experimental results of flow cytometric analysis of peripheral blood PBMC treated with a subcutaneous xenograft tumor model overexpressed in mouse MC38-ITPRIPL1-RBD;

Figure 73 shows the results of immunohistochemical staining of tumor tissue in a mouse MC38-ITPRIPL1 overexpression subcutaneous xenograft tumor model treated with a monoclonal antibody combined with ITPRIPL1-RBD;

Figure 74 shows the results of an ELISA experiment on the binding of a humanized antibody to a P8 polypeptide;

Figure 75 shows the ELISA experiment results of the binding of the corresponding polypeptide of cynomolgus monkey ITPRIPL1-P8 to the 13B7A6H3 monoclonal antibody.

Detailed ways

While this invention may be embodied in many different forms, disclosed herein are specific illustrative embodiments thereof that demonstrate the principles of the invention. It should be emphasized that the invention is not limited to the particular embodiments illustrated. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter. The experimental method that does not indicate specific condition in the following examples, generally according to conventional conditions, for example (Sambrook and Russell et al., Molecular Cloning: Laboratory Manual (Molecular Cloning-A Laboratory Manual) (Third Edition) (2001) CSHL publishes Conditions stated in the company), or in accordance with the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. Unless otherwise specified, the materials and reagents used in the examples of the present invention are all commercially available products.

Unless otherwise defined hereinafter, all technical and scientific terms used in the detailed description of the present invention have the same meanings as commonly understood by those skilled in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

The terms "comprising", "comprising", "having", "containing" or "involving" are inclusive or open-ended and do not exclude other unrecited elements or method steps. The term "consisting of is considered as a preferred embodiment of the term "comprising". If in the following a certain group is defined as comprising at least a certain number of embodiments, this is also to be understood as revealing a group which preferably consists only of these embodiments.

The use of an indefinite or definite article when referring to a noun in the singular eg "a" or "an", "the", includes a plural of that noun.

In addition, the terms first, second, third, (a), (b), (c) and the like in the specification and claims are used to distinguish similar elements and are not necessary to describe the order or time order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments described herein are capable of operation in other sequences than described or illustrated herein.

The term "and/or" is considered a specific disclosure that each of the two specified features or components is with or without the other. Thus, the term "and/or" as used herein in phrases such as "A and/or B" is intended to include A and B; A or B; A (alone); and B (alone). Likewise, the term "and/or" as used in phrases such as "A, B, and/or C" is intended to encompass each of the following: A, B, and C; A, B, or C; A, or C A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

The terms "for example" and "ie" are used by way of example only, not intended to be limiting, and should not be construed as referring to only those items explicitly recited in the specification.

The terms "or more", "at least", "more than" etc., for example "at least one" should be understood to include but not limited to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 , 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 ,87,88,89,90,91,92,93,94,95,96,97,98,99,100 or 200,300,400,500,600,700,800,900,1000,2000,3000 , 4000, 5000 or more than the stated value. Any higher numbers or fractions in between are also included.

Conversely, the term "not more than" includes every value that is less than the stated value. For example, "not exceeding 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 and 0 nucleotides. Also includes any lower numbers or fractions in between.

The terms "plurality", "at least two", "two or more", "at least a second", etc. shall be understood to include, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 , 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 ,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100 or 200,300,400,500,600,700,800,900,1000 , 2000, 3000, 4000, 5000 or more. Any higher numbers or fractions in between are also included.

The terms "approximately" and "approximately" represent the range of accuracy that can be understood by those skilled in the art and still guarantee the technical effect of the mentioned feature. The term generally means ±10%, preferably ±5%, of the indicated value.

The terms "derivative" and "mutation" refer to nucleic acid or amino acid sequence undergoing changes such as substitution, deletion, insertion, etc. to form a new sequence, and the amino acid sequence in the group composed of the new sequence has at least 70%, 80%, or 90%, 95% or 99% sequence identity;

As stated herein, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the stated range and, where appropriate, fractions thereof (e.g., tenths of an integer). one and one percent).

In order to make the purpose, technical solutions and advantages of the application clearer, some terms involved in the present invention are explained:

The term "immune response" refers to the body's defense response to foreign or mutated self components;

The term "neoplastic" means a neoplasm or solid lesion formed by abnormal cell growth;

The term "gene editing system" refers to the editing of the target gene, specifically the gene sequence obtained by means of knockout, insertion, and mutation of specific DNA fragments.

The term "sgRNA" is a guide RNA, which guides the insertion or deletion of uridine residues into the kinetoplast during the process of RNA editing, and belongs to a small non-coding RNA;

The term "antibody constant region" refers to the relatively stable region of the C-terminal amino acid of the antibody molecule, which has many important biological functions;

The term "coagulation factors" refers to various protein components involved in the blood coagulation process;

The term "antigen-presenting cells" refers to cells in the body that can ingest, process and transmit antigen information, and induce immune responses in T and B cells, mainly including macrophages, dendritic cells and B cells;

The term "autoimmune disease" refers to a disease in which the body's immune response to self-antigens results in damage to its own tissues;

The term "transplant rejection" refers to the recipient's allogeneic tissue or organ transplant when the foreign tissue or organ is recognized as a "foreign component" by the recipient's immune system, which attacks the graft , the immunological response of destruction and clearance;

The term "allergic reaction" refers to the response of tissue damage or dysfunction that occurs when the immune body is stimulated again by the same antigen;

The term "infection" refers to the local tissue and systemic inflammatory response caused by bacteria, viruses, fungi, parasites and other pathogens invading the human body;

The term "neutralizing antibody" refers to the corresponding antibody produced when pathogenic microorganisms invade the body. When pathogenic microorganisms invade cells, they need to rely on the specific molecules expressed by the pathogen itself to bind to the receptors on the cells to infect the cells and further expand. Neutralizing antibodies are certain antibodies produced by B lymphocytes, which can bind to antigens on the surface of pathogenic microorganisms, thereby preventing the pathogenic microorganisms from adhering to target cell receptors and preventing cell invasion;

The term "blocking antibody" binds to the molecular action site on the cell surface and mainly functions to block the binding of receptors and ligands;

The term "enzyme-linked immunosorbent assay" is to use the characteristic of specific bonding between antigen and antibody to detect the sample; because the antigen or antibody bound to the solid support can still have immunological activity, so the design of its bonding After the mechanism, combined with the color reaction of the enzyme, it can show whether the specific antigen or antibody exists, and can use the depth of color for quantitative analysis;

The term "flow cytometry" is used to count and sort tiny particles suspended in a fluid. This technique can be used for continuous multi-parameter analysis of individual cells flowing through optical or electronic detectors;

The term "signaling pathway" refers to the phenomenon that when a certain reaction occurs in the cell, the signal transmits a message from the outside of the cell to the inside of the cell, and the cell responds according to this message;

The term "immune escape" means that immunosuppressive pathogens antagonize, block and suppress the immune response of the body through their structural and non-structural products.

The term "modification" refers to linking other compounds or functional groups to polypeptides or proteins by means of chemical linkage, such as antibody constant region (Fc), polyethylene glycol modification, glycosylation modification, polysialic acid modification, Fatty acid modification, KLH modification, biotin modification, etc.

In this application, the term "isolated" generally refers to artificially obtained or artificially synthesized from a natural state. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of. The term "isolated" does not exclude the admixture of artificial or synthetic substances, nor the presence of other impure substances which do not affect the activity of the substance.

As used herein, the term "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds an antigen. Generally, an antibody may comprise at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding molecule thereof. Each H chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of Ab can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

The light and heavy chain variable regions each comprise a "framework" region interspersed with three hypervariable regions (also called "complementarity determining regions" or "CDRs"). A "complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" (used interchangeably herein with a hypervariable region "HVR"), is an antibody variable domain that is hypervariable in sequence and Structurally defined loops ("hypervariable loops") and/or regions containing antigen contact residues ("antigen contact points") are formed. The CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus. The CDRs located within the variable domain of an antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al, "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

However, it should be noted that the boundaries of the CDRs of the variable region of the same antibody obtained based on different assignment systems may differ. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems are different. For example, the residue ranges defined by different assignment systems for CDR regions numbered by Kabat and Chothia are shown in Table A below.

Table A. CDR residue ranges defined by different assignment systems

Thus, where reference is made to defining antibodies with a particular CDR sequence as defined in the present invention, the scope of said antibody also covers antibodies whose variable region sequences comprise said particular CDR sequence, but due to the application of a different protocol (e.g. Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.

The CDRs of the antibodies of the invention can be manually assessed to determine boundaries according to any protocol in the art or a combination thereof. Unless otherwise stated, in the present invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the above ways.

Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins , synthetic antibody, tetrameric antibody comprising two heavy chain and two light chain molecules, antibody light chain monomer, antibody heavy chain monomer, antibody light chain dimer, antibody heavy chain dimer, antibody light chain - antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugated antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv (scFv), Camelized antibodies, Affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), micro Antibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above.

The term "humanized antibody" is intended to mean an antibody obtained by grafting CDR sequences derived from the germline of another mammalian species, such as a mouse, onto human framework sequences. Additional framework region modifications can be made in the human framework sequences.

As used herein, "antigen-binding molecule", "antigen-binding fragment" or "antibody fragment" refers to any molecule comprising an antigen-binding fragment (eg, CDR) of an antibody from which the molecule is derived. Antigen binding molecules may include complementarity determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, dAbs, linear antibodies, scFv antibodies and multispecific antibodies formed from antigen binding molecules. In some embodiments, the antigen-binding molecule binds to the ITPRIPL1 protein, and in some embodiments, the antigen-binding molecule has neutralizing activity and can inhibit the binding of ITPRIPL1 to the receptor CD3E or EBI2.

The term "chimeric antigen receptor" means Chimeric Antigen Receptor (CAR), which includes an extracellular domain, a transmembrane domain and a possible intracellular domain. The extracellular domain is composed of a protein domain that recognizes and binds to a specific antigen. Chimeric antigen receptors can be expressed on immune cells and modulate their ability to interact with target cells.

The term "immunoconjugate" may include antibody immunoconjugates (i.e. antibody drug conjugates, antibody-drug conjugate, ADC), which is to link a biologically active small molecule drug to an antibody through a chemical link; similarly, Immunoconjugates also include protein-drug conjugates and nucleic acid-drug conjugates.

As used herein, the term "antigen" refers to any molecule that elicits an immune response or is capable of being bound by an antibody or antigen binding molecule. The immune response may involve antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will readily appreciate that any macromolecule, including virtually any protein or peptide, can serve as an antigen. Antigens may be expressed endogenously, ie from genomic DNA, or may be expressed recombinantly. An antigen may be specific for certain tissues, such as cancer cells, or it may be expressed broadly. In addition, fragments of larger molecules can function as antigens. In some embodiments, the antigen is an ITPRIPL1 protein antigen.

As used herein, in some embodiments, the antigen binding molecule, scFv, antibody or fragment thereof directly blocks the binding site on the ligand or alters the binding ability of the ligand through indirect means such as structural or energetic changes of the ligand . In some embodiments, the antigen binding molecule, scFv, antibody or fragment thereof prevents the biological function of the protein to which it binds.

As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound comprising amino acid residues covalently linked by peptide bonds. A protein or peptide contains at least two amino acids and there is no limit to the maximum number of amino acids that can comprise the sequence of a protein or peptide. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to short chains (which are also commonly referred to in the art as e.g. peptides, oligopeptides, and oligomers) and longer chains (which are commonly referred to in the art as proteins, which come in many types ) both. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. . Polypeptides include natural peptides, recombinant peptides, synthetic peptides or combinations thereof.

As used herein, the term "specifically binds" or "specifically binds to" refers to a non-random binding reaction between two molecules, such as between an antibody and an antigen.

As used herein, the ability to "inhibit binding", "block binding" or "compete for the same epitope" refers to the ability of an antibody to inhibit the binding of two molecules to any detectable extent. In some embodiments, an antibody that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 50%. In some embodiments, the inhibition may be greater than 20%, 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

As used herein, the term "Ka" is intended to denote the on-rate of a particular antibody-antigen interaction, while the term "Kd" as used herein is intended to denote the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "KD" or "KD value" is intended to mean the dissociation constant for a particular antibody-antigen interaction, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) and expressed as a molar concentration (M) . KD values for antibodies can be determined using methods well established in the art.

As used herein, the term "high affinity" antibody refers to an antibody with 1×10 ^-7 M or lower, more preferably 5×10 ^-8 M or lower, even more preferably 1×10 ^-8 M or lower for the target antigen. Antibodies with a KD value of low, even more preferably 5×10 ⁻⁹ M or lower, and even more preferably 1×10 ⁻⁹ M or lower.

As used herein, the term "epitope" refers to the portion of an antigen to which an immunoglobulin or antibody specifically binds. An "epitope" is also referred to as an "antigenic determinant". Epitopes or antigenic determinants usually consist of chemically active surface groups of molecules such as amino acids, carbohydrates or sugar side chains, and usually have a specific three-dimensional structure and specific charge characteristics. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontiguous amino acids in a unique three-dimensional conformation, which may be a "linear epitope" or "conformational epitope". See eg Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996). In a linear epitope, all interaction sites between a protein and an interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction sites span amino acid residues that are separated from each other in the protein. Antibodies can be screened depending on their competition for binding to the same epitope as detected by conventional techniques known to those skilled in the art. For example, competition or cross-competition studies can be performed to obtain antibodies that compete or cross-compete with each other for binding to an antigen (eg, CLDN18.2). A high-throughput method for obtaining antibodies binding to the same epitope, based on their cross-competition, is described in International Patent Application WO 03/048731.

The term "nucleic acid" or "nucleic acid sequence" in the present invention refers to any molecule, preferably a polymeric molecule, comprising ribonucleic acid, deoxyribonucleic acid or analog units thereof. The nucleic acid can be single-stranded or double-stranded. A single-stranded nucleic acid may be the nucleic acid of one strand of denatured double-stranded DNA. Alternatively, a single-stranded nucleic acid may be a single-stranded nucleic acid that is not derived from any double-stranded DNA.

The term "complementary" as used herein refers to the hydrogen bonding base pairing between the nucleotide bases G, A, T, C and U such that when two given polynucleotides or polynucleotide sequences anneal to each other In DNA, A is paired with T, G is paired with C, and in RNA, G is paired with C, and A is paired with U.

As used herein, the term "cancer" refers to a large group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth leads to the formation of malignant tumors that invade adjacent tissues and can also metastasize to remote parts of the body through the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" may include tumors. For example: bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or eye malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, gastrointestinal cancer, testicular cancer, uterine cancer, fallopian tube cancer, Endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophagus cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, Soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), childhood solid tumors, lymphocytic lymphoid tumor, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasm/tumor, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, Pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), and combinations thereof.

As used herein, the term "cancer associated with ITPRIPL1" refers to any cancer caused by, aggravated by, or otherwise associated with increased or decreased expression or activity of ITPRIPL1, and in some embodiments, the methods disclosed herein can be used to selected from colorectal cancer, lung cancer, breast cancer, melanoma, lymphoma, liver cancer, head and neck cancer, stomach cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, endometrial cancer, breast cancer, and ovarian cancer.

As used herein, an "effective dose", "effective amount" or "therapeutically effective dose" is any amount that protects a subject from onset of disease or promotes regression of disease when used alone or in combination with another therapeutic agent, said Regression of disease is evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of disease, or prevention of impairment or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be assessed using various methods known to the skilled practitioner, for example, in human subjects during clinical trials, in animal model systems for prediction of efficacy in humans, or by Activity assessment of reagents was determined in in vitro assays.

As used herein, an "individual" or "subject" is a mammal. Mammals include primates (eg, humans and non-human primates such as monkeys) and rodents (eg, mice and rats). In certain embodiments, the individual or subject is a human. A "subject" may be a "patient" - a patient is a human subject in need of treatment, may be an individual suffering from an ITPRIPL1-associated cancer, such as breast cancer, a subject at risk of developing an ITPRIPL1-associated cancer, such as breast cancer.

As used herein, the term "in vitro cell" refers to any cell cultured ex vivo. In particular, in vitro cells may include T cells.

As used herein, the term "pharmaceutically acceptable" means that the carrier, diluent, excipient and/or salt thereof is chemically and/or physically compatible with the other ingredients in the formulation and physiologically compatible with the recipient. Allow.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active agent, which is used herein are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusters, surfactants, adjuvants and ionic strength enhancers . For example, pH adjusters include but not limited to phosphate buffer; surfactants include but not limited to cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include but not limited to sodium chloride.

As used herein, the terms "regulation" and "regulation" generally include the meaning of up-regulation or down-regulation in two different directions. In some cases, it can be understood as inhibition or enhancement, and in some cases it can be understood as reduction or improvement. In some cases, it can be understood as reduction or increase, etc., and the specific interpretation is not limited, and it should be understood and interpreted according to the actual application context. Exemplarily, in some embodiments, "regulating" tumor cell growth can be understood as inhibiting or enhancing tumor cell growth.

As used herein, the terms "decrease" and "decrease" are used interchangeably and mean any change from the original. "Reduce" and "decrease" are relative terms that require a comparison between before and after measurement. "Decrease" and "decrease" include total consumption; likewise the terms "increase" and "enhance" are construed oppositely.

"Treatment" or "processing" of a subject means performing any type of intervention or procedure on a subject, or administering an active agent to a subject, in order to reverse, alleviate, ameliorate, inhibit, slow down or prevent symptoms , complications or conditions, or the onset, progression, development, severity, or recurrence of disease-related biochemical indicators. In some embodiments, "treatment" or "treatment" includes partial remission. In another embodiment, "treatment" or "treatment" includes complete remission.

The term "ITPRIPL1 expressed in an individual" refers to the expression level of ITPRIPL1 protein or mRNA in normal people and patients, including biological samples such as diseased tissues such as tumor tissues or autoimmune disease tissues, blood samples, urine, and resolution content in. This expression level can be used as a diagnostic biomarker for a disease, or as a companion diagnostic marker for related drugs.

The technical solution of the present application will be further described below in conjunction with the drawings and embodiments.

This application discloses for the first time that the extracellular region of ITPRIPL1 binds to the extracellular region of CD3ε and NRP2, and discloses the regulatory effects of ITPRIPL1 binding to CD3ε and NRP2 on the production of T cells and antigen-presenting cells, respectively. Based on new scientific discoveries, the present application discloses a method for regulating immune response and suppressing tumors by targeting ITPRIPL1, and discloses the use of regulators of ITPRIPL1 in the preparation of drugs for regulating immune response or anti-tumor. Specifically, the present application provides a method for editing the ITPRIPL1 gene, and a method for introducing genetic material into cells to regulate the expression of ITPRIPL1. This application further discloses the receptor binding domain (RBD) of ITPRIPL1 and provides a scheme for preparing ITPRIPL1-RBD isolated protein, and demonstrates the method of using ITPRIPL1-RBD to bind CD3ε and NRP2 to regulate the function of immune cells. Still further, the present application exemplifies the method for preparing ITPRIPL1 antibody, and proves that the above antibody can enhance the anti-tumor effect of immune cells. The method disclosed in the present application for regulating immune response and suppressing tumors by targeting ITPRIPL1 shows the target value of ITPRIPL1 in the treatment of tumors, autoimmune diseases, transplant rejection, allergic reactions, infections and other diseases.

In the past, there has been no report on the function of ITPRIPL1 gene. This application discloses ITPRIPL1 as a newly identified ligand for CD3ε (Example 1), and clarifies that the ITPRIPL1 extracellular region receptor binding domain (RBD) is the 25th-103th amino acid (Example 1). 2), on this basis, an isolated recombinant protein with functions of regulating CD3ε and T cells was prepared (Example 3). The application also discloses the preparation, use and administration method of the above-mentioned isolated protein.

Further, the present application discloses the ability of the isolated ITPRIPL1-RBD protein to bind to CD3ε (Example 4), respectively using enzyme-linked immunosorbent assay (ELISA), flow cytometry and other experimental methods to prove that the ITPRIPL1-RBD isolated protein can bind to CD3ε extracellular region.

Since ITPRIPL1-RBD (see SEQ ID NO:1 for the sequence) is the key region for binding CD3ε, the isolated ITPRIPL1-RBD protein can be prepared by recombinant expression, which can be used to bind to the CD3ε protein on the cell surface and deliver the inhibitory effect to T cells. Sexual signal (Example 5), so the isolated ITPRIPL1-RBD protein can be used to regulate the function of T cells. Although the application provides the sequence as the role of the isolated protein of SEQ ID NO: 1 in binding to CD3ε, regulating T cells and preparing neutralizing antibodies, non-inventive derivations (such as truncation, Other protein sequences with similar functions obtained by methods such as insertion, mutation, or fusion are also within the scope of rights claimed in this application.

This application discloses the use of isolated ITPRIPL1-RBD as an immunogen to prepare neutralizing antibodies (Example 6). In the case that ITPRIPL1 is not revealed as a ligand of CD3ε, those skilled in the art cannot purposely prepare antibodies for blocking the binding of ITPRIPL1 and CD3. This application reveals the existence of CD3ε and ITPRIPL1, a pair of receptor ligands, defines the key structure of ITPRIPL1 binding to CD3, and provides the preparation method of ITPRIPL1-RBD isolated protein, which allows the preparation of antibodies for blocking the binding of ITPRIPL1 and CD3 It becomes an easy goal for those skilled in the art to achieve through conventional techniques (such as hybridoma, phage display and other techniques). Therefore, the blocking antibody prepared by using the fragment of the extracellular region of ITPRIPL1 as an immunogen is also within the scope of the patent claims of this application.

The present application also reveals that the activation of the proliferation signaling pathway of T cell-derived cell lines is regulated by regulating the combination of ITPRIPL1-RBD and the extracellular region of CD3 (Example 7).

The present application reveals that ITPRIPL1-RBD recombinant protein can reduce the killing of human peripheral blood mononuclear cells (PBMC) to kidney-derived H3K293 cells (Example 8), and accordingly proposes the application of targeting ITPRIPL1 in inhibiting autoimmunity, and in The application value of preparing the pharmaceutical composition for treating autoimmunity, transplant rejection, allergy, infection and other diseases.

The present application reveals that reducing the expression of ITPRIPL1 on tumor cells can significantly increase the killing of human peripheral blood mononuclear cells on tumor cells (Example 9), supporting the role of ITPRIPL1 in the immune escape of tumors, and accordingly proposing ITPRIPL1 as a tumor immune system. Significant value of therapeutic targets.

The present application also revealed that the antibody prepared using ITPRIPL1-RBD protein as an immunogen effectively promoted the killing of tumor cells by immune cells (Example 10). This verifies the important value of ITPRIPL1 as a target for tumor immunotherapy, and establishes a method for blocking the combination of ITPRIPL1 and CD3 with antibodies. The antibody represents a class of antibodies with a new function, that is, an antibody that recognizes ITPRIPL1 and blocks its combination with CD3ε, thereby regulating T cell function.

Further, the present application compared ITPRIPL1 extracellular region fragments of different lengths, and proved that the ITPRIPL1-RBD2 sequence has the ability to bind to CD3ε but slightly reduced, while the ability of ITPRIPL1-RBD3 to bind CD3 was significantly reduced, thus revealing the ITPRIPL1 extracellular region The positive correlation between the length of CD3ε and the binding ability of CD3ε characterizes the basic characteristics of the functional ITPRIPL1 extracellular domain sequence (Example 11). The present application revealed that ITPRIPL1-RBD protein has the ability to bind NRP2 (Example 12). And the isolated ITPRIPL1-RBD protein has the ability to transmit inhibitory signals to differentiated THP1 macrophages expressing NRP2 protein (Example 13).

In the process of drug development, modification methods are often used to improve properties such as pharmacokinetics. Peptides or proteins can be linked to other compounds or functional groups, such as antibody constant regions (Fc), polyethylene glycol modification, glycosylation Modification, polysialic acid modification, fatty acid modification, KLH modification, biotin modification, etc. This application demonstrates the effect of modification on the recombinant protein function of ITPRIPL1 by way of Fc modification. The purified ITPRIPL1-RBD-Fc modified protein has the function of inhibiting T cell pathway signaling and killing (Example 14). Further, the ITPRIPL1-RBD-Fc protein can inhibit the phosphorylation pathway of T cells, and can be blocked by CD3ε (Example 14).

The present application prepared the Jurkat CD3 mutant and further revealed the relevant mechanism of the phosphorylation pathway (Example 15). At the same time, the influence of ITPRIPL1-Fc on Jurkat cells with different CD3 expressions was explored by detecting intracellular calcium ion flow (Example 15), and it was found that ITPRIPL1-Fc regulates the pathway by increasing the binding of CD3 and Nck (Example 16).

Further, the present application was applied to the in vivo animal model, and the MC38 subcutaneous transplanted tumor model was constructed in the humanized CD3ε mouse body, and the tumor growth, the function of T cells in PBMC, and the infiltration of T cells in tumor cells were detected (implementation Example 17). At the same time, an ITPRIPL1 knockout mouse model was established, and T cell function, cytokine changes, and testicular T cell infiltration in PBMC were detected to verify that the application could be applied in vivo (Example 17).

The present application discloses the expression of ITPRIPL1 in common multi-organ cancers and compares it with the corresponding paracancerous tissues, confirming that ITPRIPL1 is increased during canceration (Example 18).

Example 1: ITPRIPL1 as a newly identified ligand for CD3ε

1. Construction of expression plasmids

Based on the published sequence (NCBI reference sequence NM_001008949.3), the cDNA of full-length human ITPRIPL1 was synthesized, and pcDNA3.1 was used as the vector, wherein the C-terminus was fused to the Flag tag to generate the ITPRIPL1 plasmid containing the Flag tag. The results of plasmid construction are shown in Figure 1. Figure 1 (a) is a schematic diagram of the vector cloning structure, Figure 1 (b) is the DNA gel electrophoresis image of the gene expression vector plasmid after being digested by EcoRI/XhoI, and the size of the obtained fragment is in line with expectations, and Figure 1 (c) is the vector Partial interception of sequencing verification results.

2. The results of co-immunoprecipitation and immunoblotting showed that ITPRIPL1 combined with CD3.

The pcDNA3.1 plasmid containing Flag-tagged ITPRIPL1 (or empty) and HA-tagged CD3ε was co-transfected into HCT116 cells (ATCC, VA, USA) and cultured in a six-well plate (Corning, NY, USA) After 48-72 hours until the protein is fully expressed, use 1:100 immunoprecipitation lysate (Thermo Fisher, MA, USA) and protease, phosphatase, PMSF triple (Consun, Shanghai, China) to lyse and scrape the cells , a part of the cell samples were centrifuged, mixed with sample buffer (Beyond, Shanghai, China) and denatured in a metal bath at 100 degrees Celsius to obtain the input protein samples; ) for immunoprecipitation, washed with PBS, mixed with loading buffer (Beyond, Shanghai, China) and denatured in a metal bath at 100 degrees Celsius to prepare immunoprecipitated protein samples. Then configure 12.5% PAGE gel (Yase, Shanghai, China) in the gel plate (Bole, CA, USA) according to the instructions, place the prepared gel in the electrophoresis tank (Bole, CA, USA), and connect the power supply (Bole, CA, U.S.) Let the strips run through the stacking gel with a constant voltage of 80 volts, and run through the separating gel with a constant voltage of 120 volts. When the strips reached the bottom of the separating gel, transfer to the membrane using a wet method in a transfer tank (Bio-Rad, CA, USA) with a constant current of 350 mA for 90 minutes. After the membrane transfer was completed, the membrane was sheared according to the mass size of ITPRIPL1-Flag and CD3ε-HA protein. Block with fast blocking solution (Yase, Shanghai, China) for 10 minutes, and incubate ITPRIPL1-Flag and The band corresponding to CD3ε-HA was left overnight at 4°C. After washing with TBST the next day, incubate with specific anti-rabbit secondary antibody (Consun, Shanghai, China) diluted in 5% skimmed milk (Sangon, Shanghai, China) dissolved in TBS for one hour at room temperature, wash with TBST, and place Exposure to a gel imager (Bio-Rad, CA, USA) for one minute in a mixed luminescence solution (San Er, Shanghai, China).

Figure 2 shows the results of co-immunoprecipitation experiments. (a) and (b) of Figure 2 show the content of ITPRIPL1 and CD3ε in the input protein used for co-immunoprecipitation, that is, the input amount, (c) of Figure 2 shows the directly precipitated ITPRIPL1, and (d) of Figure 2 shows the indirect Precipitated CD3ε bound to ITPRIPL1. The results of co-immunoprecipitation experiments showed that ITPRIPL1 combined with CD3.

3. Immunofluorescence and colocalization analysis showed that ITPRIPL1 and CD3 had significant colocalization.

The pcDNA3.1 plasmids containing Flag-tagged ITPRIPL1 (or empty) and HA-tagged CD3ε were co-transfected into HCT116 cells (ATCC, VA, USA), placed on eight-well glass slides (Thermo Fisher, MA, USA) cultured for 30 hours until the protein was fully expressed, discarded the culture medium, washed with PBS, fixed with 4% paraformaldehyde for 20 minutes, washed again with PBS, sealed with the transmembrane blocking solution for one hour, and then added the Flag label diluted in the transmembrane blocking solution Specific mouse antibody (CST, MA, USA) was incubated with HA tag-specific rabbit antibody (CST, MA, USA) overnight at 4°C. Eight-well slides were washed overnight with PBS, and Alexa Fluor 488 fluorescent-specific anti-mouse antibody (Invitrogen, CA, U.S.) diluted in PBS was added to Alexa Fluor 594 fluorescent-specific anti-rabbit antibody (Invitrogen, CA, U.S.) to incubate at room temperature After 20 minutes, wash with PBS and mount with DAPI, and observe under a fluorescent microscope after the mounting agent is dry.

Figure 3 shows the significant intracellular co-localization of exogenously expressed ITPRIPL1 and CD3ε. Figure 3 (a) is the localization pattern of ITPRIPL1; (b) is the localization pattern of CD3ε protein; (c) is the superimposition of two color protein localization patterns, where the fluorescence intensities of ITPRIPL1 and CD3ε on the white straight path are shown in Fig. (d) of 3 shows. From the above results, it can be observed that the localization of the two proteins has a significant correlation, which supports the mutual binding of the two proteins.

Example 2: Function of ITPRIPL1 Extracellular Region Receptor Binding Domain (RBD) and Derived Sequences

1. Construction of expression plasmids

Based on the published sequence (NCBI reference sequence NM_001008949.3), the extracellular region (25-103 amino acids), extracellular region and transmembrane region (25-124 amino acids), transmembrane region and intracellular region (104- 555 amino acids) are three different target sequences, synthesize full-length human ITPRIPL1, and use pcDNA3.1 as the carrier, in which the extracellular region, the extracellular region and the C-terminus of the transmembrane region are fused to the Flag tag and green fluorescent protein (GFP), The N-terminus of the transmembrane region and the intracellular region is fused to the Flag tag and green fluorescent protein (GFP), thereby generating the ITPRIPL1 extracellular region, extracellular region, transmembrane region, and transmembrane region containing the Flag tag and green fluorescent protein (GFP) and intracellular domain plasmids. Among them, the result of constructing the expression vector of ITPRIPL1(25-103), which is the CD3 binding domain, is shown in FIG. 4 . (a) of Figure 4 is the vector plasmid construction map, the cDNA encoding the ITPRIPL1 (25-103) amino acid fragment is linked to the cDNA encoding the Flag tag at the end, and inserted into pEGFP-C1 (Shanghai Jierui Company), the vector expresses the product It is fluorescently labeled with GFP, but its main functional product is ITPRIPL1 (25-103), which is a CD3 binding fragment. Figure 4 (b) is the DNA gel electrophoresis image after the gene expression vector plasmid was digested by EcoRI/XhoI, and the size of the obtained fragments is in line with expectations, and Figure 4 (c) is a screenshot of the peak diagram of the sequencing result.

2. The results of co-immunoprecipitation experiments showed that the extracellular region of ITPRIPL1 (rather than the intracellular region or transmembrane region) combined with CD3ε.

The pcDNA3.1 plasmid containing Flag-tagged ITPRIPL1 extracellular region, extracellular region and transmembrane region, intracellular region and transmembrane region, and HA-tagged CD3ε were co-transfected into HCT116 cells (ATCC, VA, USA) , placed in a six-well plate (Corning, NY, U.S.) and cultured for 48-72 hours until the protein was fully expressed, then immunoprecipitated lysate (Thermo Fisher, MA, U.S.) was mixed with protease, phosphatase, and PMSF at a ratio of 1:100 ( Kangchen, Shanghai, China) mixed lysate to lyse and scrape the cells, a part of the cell samples were centrifuged, mixed with loading buffer (Biyuntian, Shanghai, China) and denatured in a metal bath at 100 degrees Celsius to obtain input protein samples; the remaining cells The sample was immunoprecipitated with a Flag-tag-specific mouse antibody (CST, MA, USA), washed with PBS, mixed with a loading buffer (Beyond, Shanghai, China) and denatured in a metal bath at 100 degrees Celsius to prepare an immunoprecipitated protein sample. Then configure 12.5% PAGE gel (Yase, Shanghai, China) in the gel plate (Bole, CA, USA) according to the instructions, place the prepared gel in the electrophoresis tank (Bole, CA, USA), and connect the power supply (Bole, CA, U.S.) Let the strips run through the stacking gel with a constant voltage of 80 volts, and run through the separating gel with a constant voltage of 120 volts. When the strips reached the bottom of the separating gel, transfer the membrane using the wet method, and transfer the membrane at a constant current of 350 mA for 90 minutes in a transfer tank (Bio-Rad, CA, USA). After the membrane transfer is completed, the membrane shearing process is carried out according to the mass size of ITPRIPL1 extracellular region, extracellular region and transmembrane region, intracellular region and transmembrane region-Flag and CD3ε-HA protein. Block with fast blocking solution (Yase, Shanghai, China) for 10 minutes, and incubate ITPRIPL1-Flag and The band corresponding to CD3ε-HA was left overnight at 4°C. After washing with TBST the next day, incubate with specific anti-rabbit secondary antibody (Consun, Shanghai, China) diluted in 5% skimmed milk powder (Sangon, Shanghai, China) dissolved in TBS for one hour at room temperature, wash with TBST, and place Exposure to a gel imager (Bio-Rad, CA, USA) for one minute in a mixed luminescence solution (San Er, Shanghai, China).

Figure 5 shows the results of co-immunoprecipitation experiments. As shown in Figure 5, (a) of Figure 5 shows the content of ITPRIPL1 in the input protein detected by the Flag antibody, and Figure 5 (b) shows the content of CD3ε in the input protein. Figure 5(c) shows directly precipitated ITPRIPL1 different mutants, while Figure 5(d) shows the corresponding CD3ε that was indirectly precipitated (due to binding to ITPRIPL1 mutants). The above results suggest that CD3ε and ITPRIPL1 remain bound in the presence of the extracellular domain of ITPRIPL1 (rather than the intracellular or transmembrane domain). Therefore, the extracellular region of ITPRIPL1 binds to CD3ε, which is consistent with the conclusion that the receptor binding domain (RBD) of the extracellular region of ITPRIPL1, a ligand of CD3ε, is amino acids 25-103.

3. Immunofluorescence and co-localization analysis showed that ITPRIPL1 extracellular domain and CD3 extracellular domain had trans binding.

HA-tagged CD3ε was transfected into HCT116 cells; in addition, Flag-tagged ITPRIPL1 (respectively, human, mouse, gorilla, green monkey, golden snub-nosed monkey, Amazon squirrel monkey ITPRIPL1 extracellular region and human ITPRIPL1 transmembrane region )-green fluorescent protein pcDNA3.1 plasmid was transfected into another batch of HCT116; finally, the two transfected cells were co-cultured, and the localization patterns of the two proteins were determined by immunofluorescence double staining. The extracellular region sequences of ITPRIPL1 of rat, mouse, gorilla, green monkey, golden snub-nosed monkey, black snub-nosed monkey, and Amazon squirrel monkey are shown in FIG. 54 .

The specific steps are as follows: Transfect a batch of HCT116 cells (ATCC, VA, USA) with pcDNA3.1 plasmid containing HA-tagged CD3ε; in addition, separate ITPRIPL1 (extracellular region and transmembrane region)-green fluorescent protein containing Flag tag The pcDNA3.1 plasmid was used to transfect another batch of HCT116 cells (ATCC, VA, the United States); 20 hours after transfection, the cells were digested with trypsin, mixed and fully resuspended, and plated onto eight-well glass slides (Sai Murphy, MA, USA), and the culture was continued for 10 hours. Discard the culture medium, wash with PBS, fix with 4% paraformaldehyde for 20 minutes, wash with PBS again, and seal with the permeable blocking solution for one hour, then add the HA tag-specific rabbit antibody (CST, MA, USA) diluted in the permeable blocking solution. ) and incubated overnight at 4°C. Eight-well slides were washed overnight with PBS, and Alexa Fluor594 fluorescent-specific anti-rabbit antibody (Invitrogen, CA, USA) diluted in PBS was added to incubate at room temperature for 20 minutes. Observed under a fluorescence microscope. The localization patterns of the two proteins were determined by immunofluorescence double staining.

As shown in Figure 6, (a) of Figure 6 shows the overlapping images of ITPRIPL1 and CD3ε staining, and (b) and (c) of Figure 6 show the staining results of the ITPRIPL1 receptor binding region (ITPRIPL1-RBD) and CD3ε, respectively. Figure 6(d) is the colocalization region of the two proteins obtained by analyzing the colocalization analysis module of the ImageJ software package. The fluorescence intensities of ITPRIPL1 and CD3ε on the white straight path are shown in (e) of FIG. 6 . It can be seen that at the junction of the two cells, the signals of ITP-RBD (green fluorescence) and CD3ε (red fluorescence) are significantly concentrated and strengthened, which supports the trans-binding of the extracellular regions of the two proteins on the cell surface. The experimental results also showed that the extracellular region of ITPRIPL1 in mice, gorillas, green monkeys, golden snub-nosed monkeys, and Amazon squirrel monkeys combined with the extracellular region of human CD3E.

Example 3: Preparation of ITPRIPL1-RBD isolated recombinant protein with regulation of CD3ε and T cell functions

Based on the published sequence (NCBI reference sequence NP_001008949.1), the recombinant protein of human ITPRIPL1 extracellular region (25-103 amino acids) was synthesized by Cusabio (Wuhan, China), expressed and purified by yeast, in which the C-terminus was fused to 6x-His and Myc tag to produce ITPRIPL1-RBD recombinant protein containing 6x-His and Myc tag.

As shown in Figure 7, it is the result of Coomassie brilliant blue staining after gel electrophoresis of ITPRIPL1-RBD recombinant protein, suggesting that its molecular weight and purity meet expectations.

Example 4: Concanavalin A (ConA) and the isolated ITPRIPL1-RBD protein have the ability to bind CD3ε respectively

Enzyme-linked immunosorbent assay (ELISA) confirmed concanavalin A (ConA) and isolated ITPRIPL1 extracellular region fragments had concentration-dependent direct binding to the extracellular region of CD3ε. A special plate for ELISA (costar, ME, USA) was used. First, 0.5/1/2/4 micrograms per milliliter of ConA or 0.03125/0.0625/0.125/0.25/0.5/1/2 micrograms per milliliter of ITPRIPL1-RBD recombinant protein were dissolved in 100 microliters of ELISA coating solution (Solarbio, Beijing, China) and the negative control was coated with 100 microliters of coating solution without protein. Four times wrapped overnight. After washing with PBST, 100 microliters of 5% BSA dissolved in PBS (VWR, PA, USA) was used to block, and the incubator was blocked at 37 degrees for 90 minutes. After washing with PBST, incubate and bind with 1 microgram per milliliter of CD3ε extracellular region protein fragments (Shenzhou, Beijing, China), the CD3ε (Met1-Asp126) protein has hFc tag, and bind at 37 degrees for 60 minutes . After washing with PBST, specific anti-human Fc fragment antibody (Abcam, MA, USA) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, configure chromogenic solution (Sangon, Shanghai, China), 100 microliters per well, and place in an incubator for 5-30 minutes to react, then add 50 microliters of stop solution (Sangon, Shanghai, China), and set The color was read at 450 nm on a microplate reader (Thermo Fisher, MA, USA).

Fig. 8 is the result of ELISA experiment. Experiments showed that concanavalin A (ConA) and isolated ITPRIPL1 extracellular domain purified protein (IT1-RBD) had concentration-dependent direct binding to CD3 extracellular domain purified protein respectively, and the binding strength of IT1-RBD was greater than that of ConA. The above results prove that concanavalin A (ConA) and the isolated purified extracellular region of ITPRIPL1 (IT1-RBD) directly bind to the purified protein of the extracellular region of CD3ε, and the binding strength of IT1-RBD is greater than that of ConA.

Example 5: The isolated ITPRIPL1-RBD protein can be used to bind to the CD3ε protein on the cell surface and transmit inhibitory signals to T cells

1. Construction of HCT116-ITPRIPL1 and HCT116-CD3ε stable transgenic cells

The constructed full-length ITPRIPL1-Flag plasmid, CD3ε-HA plasmid and empty pcDNA3.1 plasmid were respectively transfected into HCT116 cells (ATCC, VA, USA), placed in an incubator for 24-48 hours, and then added 1000 μg Every milliliter of geneticin (Geneticin, G418) (Gibco, CA, U.S.) was screened, and after 10-14 days to the death of all the cells in the empty pcDNA3.1 plasmid transfection group, the HCT116-ITPRIPL1 and HCT116-CD3ε stable cells were obtained. Transplanted cells.

2. Flow cytometry proved that the purified protein fragment of the extracellular region of ITPRIPL1 binds to Jurkat cells with high expression of CD3.

The cultured Jurkat cells (ATCC, VA, USA) were counted, the cell number was adjusted to 2×10 ⁵ per milliliter, and 200 microliters were added to six 1.5ml EP tubes (Axygen, CA, USA). Add 0.1 micrograms, 0.2 micrograms, 0.4 micrograms, and 0.8 micrograms of ITPRIPL1-RBD-6x-His recombinant protein (IT1-6x-His protein) to four of the EP tubes to make the concentrations 0.5 micrograms per milliliter and 1 microgram per milliliter , 2 micrograms per milliliter, 4 micrograms per milliliter. Place all the EP tubes in the cell culture incubator, let them stand for 30 minutes, then take out the EP tubes, centrifuge at 400 rcf for 5 minutes, and discard the supernatant. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded again and the washing was repeated. Dilute 6x-His-FITC antibody (Abcam, MA, USA) 1:500 with cell staining buffer, add 200 microliters of antibody diluent to each EP tube except the negative control, and incubate at room temperature on a shaker at 40 rpm for 30 minutes . Then take out the EP tube, centrifuge at 400 rcf for 5 minutes and discard the supernatant. Wash the cells by resuspending them with 1000 μl of cell staining buffer, centrifuge at 400 rcf for 5 minutes, discard the supernatant again and repeat the washing. After washing, each EP tube was resuspended by adding 300 microliters of cell staining buffer, and transferred to a flow tube (Falcon, NY, USA) for analysis on a machine (Miltenyi, Cologne, Germany).

(a) of FIG. 9 is the threshold setting for not classifying dead cells. (b) of FIG. 9 reflects the binding status of Jurkat cells to different concentrations of purified protein fragments of the extracellular region of ITPRIPL1. Figure 10 shows the specific staining and protein binding conditions under each condition in (b) of Figure 9 . The above results indicated that the purified protein fragment of ITPRIPL1 extracellular region could bind to Jurkat cells with high expression of CD3.

3. Flow cytometry proves that ITPRIPL1 protein binds to cells overexpressing CD3 with higher efficiency.

Digest and count the cultured HCT116 wild-type cells (ATCC, VA, USA) and HCT116-CD3ε stable transfection cells, adjust the cell number to ^2x105 per milliliter, add 200 microliters to 5 and 3 1.5ml EP respectively tube (Axygen, CA, USA). Add 0.2 μg, 0.4 μg, and 0.8 μg of ITPRIPL1-RBD-6x-His recombinant protein to the 3 EP tubes of HCT116 wild cells and the 3 EP tubes of HCT116-CD3ε stable transfection cells respectively to make the concentration of 1 μg per milliliters, 2 micrograms per milliliter, 4 micrograms per milliliter. Place all the EP tubes in the cell culture incubator, let them stand for 30 minutes, then take out the EP tubes, centrifuge at 400 rcf for 5 minutes, and discard the supernatant. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded again and the washing was repeated. Dilute 6x-His-FITC antibody (Abcam, MA, USA) 1:500 with cell staining buffer, add 200 microliters of antibody diluent to each EP tube except the negative control, and incubate at room temperature on a shaker at 40 rpm for 30 minutes . Then take out the EP tube, centrifuge at 400 rcf for 5 minutes and discard the supernatant. Wash the cells by resuspending them with 1000 μl of cell staining buffer, centrifuge at 400 rcf for 5 minutes, discard the supernatant again and repeat the washing. After washing, each EP tube was resuspended by adding 300 microliters of cell staining buffer, and transferred to a flow tube (Falcon, NY, USA) for analysis on a machine (Miltenyi, Cologne, Germany).

(a) of FIG. 11 is the threshold setting for not including dead cells. (b) of FIG. 11 reflects the combination of cells with different CD3ε expression and different concentrations of ITPRIPL1 recombinant protein. Figure 12 shows the specific staining and protein binding conditions under each condition in (b) of Figure 11 . The above results indicated that the purified protein fragment of the extracellular region of ITPRIPL1 could bind to HCT116 cells overexpressing CD3 but not to HCT116 cells not expressing CD3.

4. Flow cytometry demonstrated that CD3 binds to cells overexpressing ITPRIPL1 with higher efficiency.

Digest and count the cultured HCT116 wild-type cells (ATCC, VA, USA) and HCT116-ITPRIPL1 stable transfection cells, adjust the number of cells to ^2x105 per milliliter, add 200 microliters to three and one 1.5ml EP respectively tube (Axygen, CA, USA). Add 0.4 micrograms of CD3E-human Fc protein (Shenzhou, Beijing, China) to an EP tube of HCT116 wild cells and an EP tube of HCT116-ITPRIPL1 stable cells to make the concentration 2 micrograms per milliliter. Place all the EP tubes in the cell culture incubator, let them stand for 30 minutes, then take out the EP tubes, centrifuge at 400 rcf for 5 minutes, and discard the supernatant. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded again and the washing was repeated. Dilute anti-human IgG Alexa Fluor 647 antibody (Invitrogen, CA, USA) 1:1000 with cell staining buffer, add 200 microliters of antibody diluent to each EP tube except the negative control, and incubate at room temperature on a shaker at 40 rpm for 30 minute. Then take out the EP tube, centrifuge at 400 rcf for 5 minutes and discard the supernatant. Wash the cells by resuspending them with 1000 μl of cell staining buffer, centrifuge at 400 rcf for 5 minutes, discard the supernatant again and repeat the washing. After washing, each EP tube was resuspended by adding 300 microliters of cell staining buffer, and transferred to a flow tube (Falcon, NY, USA) for analysis on a machine (Miltenyi, Cologne, Germany).

(a) of FIG. 13 is the threshold setting for not classifying dead cells. (b) of FIG. 13 reflects the binding status of cells with different expression status of ITPRIPL1 to CD3ε protein. Figure 14 shows the specific staining and protein binding conditions under each condition in (b) of Figure 13 . The above results indicated that CD3ε could bind to HCT116 expressing ITPRIPL1, and could bind more strongly to HCT116 cells overexpressing ITPRIPL1.

5. The luciferin reporter experiment proved that the purified protein fragment of the extracellular region of ITPRIPL1 inhibited the proliferation signal of NFKB in Jurkat-dual cells activated by ConA.

The cultured Jurkat-dual cells (Invivogen, CA, U.S.) were counted, centrifuged at 1000 rpm for 5 minutes and then resuspended with antibiotic-free IMDM medium (Gibco, CA, U.S.) to adjust the number of cells to ^2x10 per milliliter, transparent 96-well 200 microliters of cells were added to each well of the plate (Thermo Fisher, MA, USA). 0.2 micrograms, 0.4 micrograms, and 0.8 micrograms of ITPRIPL1-6x-His recombinant protein were added to each well so that the concentrations were 1 microgram per milliliter, 2 micrograms per milliliter, and 4 micrograms per milliliter. After two hours of reaction in a cell culture incubator, 10 micrograms per milliliter of concanavalin A (ConA) (Aladdin, Shanghai, China) was added to each well, and placed in a cell culture incubator for 18-24 hours of reaction. After the reaction is completed, take an opaque 96-well plate (costar, ME, USA), add 50 microliters of Quanti-luc reagent (Invivogen, CA, USA) and 20 microliters of reaction mixture to each well, add and mix well, and then use the multifunctional enzyme A marker (Thermo Fisher, MA, USA) immediately detects the signal.

As shown in FIG. 15 , the NFKB signal changes with the ITPRIPL1 protein concentration under the activation condition of 10 micrograms per milliliter of ConA. The above results indicated that the purified recombinant protein of ITPRIPL1-RBD could inhibit the NFKB proliferation signal of ConA-activated Jurkat-dual cells in a concentration-dependent manner.

6. The luciferin reporter experiment proved that the purified protein fragment of the extracellular region of ITPRIPL1 immobilized on the surface of the microsphere inhibited the proliferation signal of NFKB in Jurkat-dual cells activated by ConA.

Protein G magnetic microspheres (Thermo Fisher, MA, U.S.) and His antibody (Abcam, MA, U.S.) were placed in 4 EP tubes (Axygen, CA, U.S.), and put into a DNA mixer (Xinzhi, Ningbo, China). , China) at the lowest speed for 1 hour at room temperature. Add 0.2 μg, 0.4 μg, and 0.8 μg of ITPRIPL1-6x-His recombinant protein to three of the EP tubes, and rotate at room temperature at the lowest speed for 1 hour again. Cultured Jurkat-dual cells (Invivogen, CA, U.S.) were counted, centrifuged at 1000rpm for 5 minutes and then resuspended in antibiotic-free IMDM medium (Gibco, CA, U.S.) to adjust the number of cells to ^2x106 per milliliter, transparent 96-well plate (Thermo Fisher, MA, USA) was added 200 microliters of cells per well. The contents of each EP tube were added to each well to make the coating protein concentration 1 microgram per milliliter, 2 microgram per milliliter, and 4 microgram per milliliter. After two hours of reaction in a cell culture incubator, 50 micrograms per milliliter of concanavalin A (ConA) (Aladdin, Shanghai, China) was added to each well, and placed in a cell culture incubator for 18-24 hours of reaction. After the reaction is completed, take an opaque 96-well plate (costar, ME, USA), add 50 microliters of Quanti-luc reagent (Invivogen, CA, USA) and 20 microliters of reaction mixture to each well, add and mix well, and then use the multifunctional enzyme A marker (Thermo Fisher, MA, USA) immediately detects the signal.

As shown in Figure 16, under the condition of 50 micrograms per milliliter of ConA activation, the NFKB signal changes with the change of the concentration of ITPRIPL1 protein coated on the microspheres. The above results indicated that the purified recombinant ITPRIPL1-RBD protein immobilized on the surface of microspheres could inhibit the NFKB proliferation signal of ConA-activated Jurkat-dual cells in a concentration-dependent manner.

7. Detect the expression level of ITPRIPL1 in different types of tumor cells

Since the extracellular region of ITPRIPL1 has the function of suppressing immune cells (such as T cells), tumor cells may express ITPRIPL1 to escape the surveillance and killing of the immune system. If a variety of tumor cells abnormally express ITPRIPL1, it is suggested that ITPRIPL1 may play a role in promoting the occurrence and development of tumors (including immune escape). The present invention reveals that ITPRIPL1 is abnormally expressed in cell lines from different types of tumors. The specific experimental steps are as follows: count the cultured cell lines, take ^2x106 cells into a 15ml centrifuge tube, centrifuge at 800rpm x 4 minutes, discard the supernatant, resuspend and wash in PBS, centrifuge at 800rpm x 4 minutes, and repeat the resuspension and washing in PBS. Discard the supernatant after centrifugation, and prepare RIPA lysate, protease, phosphatase, and PMSF at a ratio of 1:100. Add 120 microliters of the mixed lysate to each tube of cells and transfer it to an EP tube. Each EP tube was frozen three times and thawed three times on liquid nitrogen-ice, and centrifuged at 12000 rpm at 4 degrees Celsius for 15 minutes after the last thaw. After the centrifugation, take the supernatant. After centrifugation, the supernatant: 5x sample buffer is configured at a ratio of 4:1 to form each cell sample, and placed in a metal bath at 100 degrees Celsius for 10 minutes to denature. After denaturation was completed, the endogenous expression of ITPRIPL1 in each tumor cell line was determined by gel electrophoresis and immunoblotting analysis, using GAPDH as an internal reference.

Figure 17 shows the expression of ITPRIPL1 after adjusting the internal reference of GAPDH in Western blotting. As shown in Figure 17, immunoblotting experiments detected the expression level of ITPRIPL1 protein in tumor cell lines, including LoVo colorectal cancer, Raji lymphoma, RL lymphoma, MDA-MB-231 breast cancer, HCT116 colorectal cancer, A549 lung cancer, HL60, Jurkat lymphoma, H1299 lung cancer, A375 melanoma cells all have higher levels of expression. Although the inventors have not detected the expression of ITPRIPL1 in all types of tumor cells, judging from the ratio of ITPRIPL1 expression in the tumor cells that have been detected (10/11, more than 90%), ITPRIPL1 may exist in a variety of malignant tumors. type, a fairly wide range of expressions.

Correspondingly, the results extracted from the Protein Atlas database based on high-throughput mRNA expression profiling analysis suggest that ITPRIPL1 may have cases of significantly increased expression in various tumors. We haven't seen any report on the function of ITPRIPL1 in the past. Therefore, before the disclosure of the present invention, those skilled in the art cannot predict any function of ITPRIPL1 in tumorigenesis and immune escape. The present invention discloses experimental data such as abnormal expression of ITPRIPL1 tumor cell protein level increase, ITPRIPL1 extracellular region binding to CD3ε and causing T cell inhibition, which reveals the important value of ITPRIPL1 in tumor immunotherapy targets for the first time.

As shown in Figure 18, according to the mRNA expression profile data collected on the Protein Atlas website, ITPRIPL1 may be mainly expressed in testis and T cells in normal tissues or cells. It is worth noting that the expression of mRNA does not directly represent the expression at the protein level, and in general, the reanalysis of expression profile data needs to be verified by low-throughput biological experiments (such as protein gel electrophoresis and western blot analysis) to draw reliable conclusions.

8. The OCTET molecular interaction experiment proves that the purified protein fragment of the extracellular region of ITPRIPL1 can directly bind to CD3ε protein.

Start the OCTET instrument. 100 micrograms of CD3E-Fc protein (Shenzhou, Beijing, China) was adsorbed to saturation by the Fc probe, and then 50 micrograms of ITPRIPL1 extracellular region recombinant protein were combined at the concentration of 800nM/1600nM/3200nM, the binding curve was drawn and Calculate the dissociation constant.

As shown in Fig. 40, the figure shows the whole adsorption-dissociation process and calculates the relevant constants. The above results indicated that the purified protein fragment of the extracellular region of ITPRIPL1 could directly bind to CD3E protein.

Example 6: The isolated ITPRIPL1-RBD is used as an immunogen to prepare antibodies for inhibiting tumor growth in vivo

1. ITPRIPL1-RBD was used as immunogen to prepare mouse polyclonal antibody and polyclonal antibody

Human ITPRIPL1-RBD recombinant protein was used as the immunogen, and after the purity and biological activity of the sample were verified in the above examples, C57BL/6 mice were immunized, and multiple immunizations were performed to enhance the effect: (1) Initial immunization , antigen 50 μg/rat, with Freund’s complete adjuvant subcutaneously multi-point injection, with an interval of 3 weeks; (2) the second immunization, the dosage route is the same as above, with Freund’s incomplete adjuvant, with an interval of 3 weeks; (3) The third immunization , the dose is the same as above, without adjuvant, intraperitoneal injection at intervals of 3 weeks; (4) booster immunization, dose 50 μg, intraperitoneal injection. Blood was collected 3 days after the last injection to measure its potency. After testing the immune effect to meet the requirements, blood was collected multiple times, the polyclonal antibody was separated, and the polyclonal antibody was purified. The specific experimental steps included: (1) Prepare protein G sepharose CL-4B affinity column. Prepare 10mL of protein G sepharose CL-4B filler, mix equal volumes of filler and TBS buffer solution in a vacuum bottle, and stir. Vacuum for 15 minutes to remove air bubbles in the filling. Slowly add the protein G sepharose CL-4B filler into the glass column, use the pump to control the filling speed to 1mL/min-2mL/min, avoid column drying, and use 10 times the bed volume and pre-cooled TBS buffer solution to balance the column. (2) Preparation of polyclonal antibodies. Thaw the polyclonal antibody slowly in ice water or in a 4°C refrigerator to avoid protein aggregation. Aggregations that occur during protein thawing can be dissolved by preheating at 37°C. Add solid sodium azide to a concentration of 0.05%, centrifuge at 15,000×g for 5 minutes at 4°C, remove the clarified polyclonal antibody and filter it to remove excess fat. (3) Affinity chromatography. Dilute the antibody with TBS buffer solution at a ratio of 1:5, and then filter through a filter. Put the polyclonal antibody onto the column at a rate of 0.5 mL per minute. In order to ensure the combination of the polyclonal antibody and the filler, it is necessary to load the column twice continuously and keep the sample effluent. Wash the column with TBS buffer solution until Aλ280nm<0.008, then add Ph 2.7 elution buffer solution, and elute at a speed of 0.5mL/min until all proteins flow down. Collect the eluate with a 1.5mL EP tube that has been added with 100 μL of neutralizing buffer solution. After mixing, check the pH of the eluent with pH test paper. If the pH is lower than 7, use the neutralizing buffer to adjust it to about pH 7.4. Prevent denaturation of antibodies. Add 10 mL of pH 1.9 elution buffer solution to the column, and collect the eluate as described above until Aλ280nm<0.008. The protein content in each tube was determined using a spectrophotometer.

2. Enzyme-linked immunosorbent assay (ELISA) proved that polyclonal antibody against ITPRIPL1-RBD as immunogen can bind to cells expressing ITPRIPL1

A special plate for ELISA (costar, ME, USA) was used. First, B16 cells that do not express ITPRIPL1 (ATCC, VA, the United States), LoVo cells that express ITPRIPL1 to a moderate extent (ATCC, VA, the United States) and HCT116-ITPRIPL1 stably transfected cells were trypsinized and counted, and the number of cells was adjusted to 2x106 Cells were plated at 100 microliters per well per milliliter and coated overnight at 4°C. After washing with PBST, 100 microliters of 5% skimmed milk powder dissolved in PBS (Sangon, Shanghai, China) was used for blocking, and the incubator was blocked for 90 minutes at 37°C. After washing with PBST, the polyclonal antibody with a total IgG concentration of 10 mg/mL was serially diluted at 1:1000/1:500/1:250/1:125, and incubated with the plated cells for binding at 37 degrees in an incubator for 60 minute. After washing with PBST, specific anti-mouse Fc fragment antibody (Consun, Shanghai, China) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, configure chromogenic solution (Sangon, Shanghai, China), 100 microliters per well, and place in an incubator for 5-30 minutes to react, then add 50 microliters of stop solution (Sangon, Shanghai, China), and set The color was read at 450 nm on a microplate reader (Thermo Fisher, MA, USA).

(a) of FIG. 19 shows the changes in the binding rate of B16 cells to different concentrations of polyclonal antibodies. (b) of FIG. 19 shows the change of binding rate between LoVo cells and different concentrations of polyclonal antibodies. (c) of FIG. 19 shows the changes in the binding rate of HCT116-ITPRIPL1 stable transfected cells to polyclonal antibodies at different concentrations. The experiment showed that with the increase of IgG concentration in the polyclonal antibody, the binding rate of B16 cells and polyclonal antibody did not change significantly, but the binding rate of LoVo cells and polyclonal antibody showed a concentration-dependent increase to a certain extent, HCT116-ITPRIPL1 stable transfection strain The binding rate of cells to polyclonal antibody showed a significant concentration-dependent increase. The above results demonstrate that polyclonal antibodies can bind to cells expressing ITPRIPL1.

3. Enzyme-linked immunosorbent assay (ELISA) proved that the polyclonal antibody could block the combination of ITPRIPL1 and CD3ε.

A special plate for ELISA (costar, ME, USA) was used. First, 0.1 microgram of CD3ε protein fragment (Shenzhou, Beijing, China) was dissolved in 100 microliters of ELISA coating solution (Solarbio, Beijing, China) to coat the plate. The CD3ε (Met 1-Asp117) protein with hFc tag was negative The control was coated with 100 microliters of coating solution without protein. Coating overnight at 4°C. After washing with PBST, 100 microliters of 5% skimmed milk powder dissolved in PBS (Sangon, Shanghai, China) was used for blocking, and the incubator was blocked for 90 minutes at 37°C. After washing with PBST, 2 micrograms per milliliter of ITPRIPL1-RBD-6x-His protein was mixed with 1:1000/1:500/1:250/1:125 polyclonal antibodies with a total IgG concentration of 10 mg/ml Afterwards, co-incubation and combination were carried out, and the incubator was combined at 37 degrees for 60 minutes. After washing with PBST, specific anti-6x-His tag horseradish peroxidase antibody (Abcam, MA, USA) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, configure chromogenic solution (Sangon, Shanghai, China), 100 microliters per well, and place in an incubator for 5-30 minutes to react, then add 50 microliters of stop solution (Sangon, Shanghai, China), and set The color was read at 450 nm on a microplate reader (Thermo Fisher, MA, USA).

As shown in Figure 20, this experiment shows that with the increase of IgG concentration in the polyclonal antibody, the binding rate of CD3ε to ITPRIPL1-RBD protein gradually decreases. The above results prove that polyclonal antibodies can block the binding between ITPRIPL1 and CD3ε.

4. Flow cytometry proved that polyclonal antibody can block the binding of ITPRIPL1 to cells overexpressing CD3ε.

The cells of HCT116-CD3ε stable transfection strain were digested and counted, the cell number was adjusted to 2×10 ⁵ per milliliter, and 200 microliters were added to eight 1.5ml EP tubes (Axygen, CA, USA). Add 0.8 micrograms of IT1-RBD protein to each of the EP tubes to make the concentration 4 micrograms per milliliter. Add 1:1000/1:500/1:250/1:125/1:67.5 diluted polyclonal antibody with a total IgG concentration of 10 mg/ml to five EP tubes, and let stand in the cell culture incubator for 30 minutes . Then take out the EP tube, centrifuge at 400 rcf for 5 minutes and discard the supernatant. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded again and the washing was repeated. Dilute 6x-His FITC antibody (Abcam, MA, USA) at 1:500 with cell staining buffer, add 200 microliters of antibody diluent to each EP tube except the negative control, and incubate at room temperature on a shaker at 40 rpm for 30 minutes. Take out the EP tube, centrifuge at 400rcf for 5 minutes and discard the supernatant. Wash the cells by resuspending them with 1000 μl of cell staining buffer, centrifuge at 400 rcf for 5 minutes, discard the supernatant again and repeat the washing. After washing, each EP tube was resuspended by adding 300 microliters of cell staining buffer, and transferred to a flow tube (Falcon, NY, USA) for analysis on a machine (Miltenyi, Cologne, Germany).

(a) of FIG. 21 is the threshold setting for not classifying dead cells. (b) of Figure 21 reflects the changes in the binding between ITPRIPL1 and CD3ε when the concentration of the polyclonal antibody changes. Figure 22 shows the specific staining and protein binding conditions under each condition in (b) of Figure 21. The above results indicated that the polyclonal antibody could block the binding of ITPRIPL1 to cells overexpressing CD3ε.

5. Prepare monoclonal antibodies on the basis of polyclonal antibodies, and further verify the effect of antibodies on inhibiting tumor growth.

Since ITRPRIPL1 is upregulated in a variety of tumors, antibodies that specifically bind to ITPRIPL1 can recognize tumor cells in the body and kill tumor cells by exerting ADCC, ADCP and CDC functions through the Fc segment of the constant region of the antibody. ADCC is Antibody-Dependent Cell-mediated Cytotoxicity (Antibody-Dependent Cell-mediated Cytotoxicity), which means that the Fab segment of the antibody binds to the epitope of virus-infected cells or tumor cells, and its Fc segment interacts with killer cells (NK cells) , macrophages, neutrophils, etc.) FcR binding on the surface, mediating killer cells directly kill target cells, is an important mechanism of action of anti-tumor therapeutic antibody drugs. ADCP, or Antibody-Dependent Cellular Phagocytosis, is also an important mechanism for identifying and mediating the action of therapeutic antibodies on tumor cells. CDC is complement-dependent cytotoxicity, which refers to the cytotoxicity involved in complement, that is, through the binding of specific antibodies to the corresponding antigens on the cell membrane surface, a complex is formed to activate the classic pathway of complement, and the formed membrane attack complex exerts its effect on target cells. cracking effect. This example will demonstrate the inhibitory effect of the antibody that specifically recognizes the protected region of ITPRIPL1 on tumor growth in vivo. The specific implementation steps are as follows:

a) Dilute the fused hybridoma cells into a 96-well plate (estimated density is 0.5 cells per well), and further culture until colony formation. The medium supernatant derived from monoclonal hybridoma was taken for ELISA test, and the degree of antigen binding to 0.2 micrograms per milliliter of ITPRIPL1 (RBD1 protein) was determined, sorted according to the OD450 absorption value, and the antibody corresponding to the strongest binding was taken The monoclonal hybridoma cells were expanded and cultivated, and the ascites antibody (hereinafter referred to as: RBD1-binding antibody) was prepared for functional research in animals.

b) The constructed full-length ITPRIPL1-Flag plasmid and the empty pcDNA3.1 plasmid were transfected into MC38 cells (Kerafast, MA, USA), placed in an incubator for 24-48 hours, and then added 200 micrograms per milliliter of Geneticin (G418) (Gibco, CA, USA) was used for selection, and MC38-ITPRIPL1 stably transfected cells were obtained after 10-14 days until all the cells in the empty pcDNA3.1 plasmid transfected group died. Humanized CD3ε mice aged 6-8 weeks (Southern model, Shanghai, China) were randomly divided into groups according to body weight, with 6 mice in each group. Count the MC38 wild-type and MC38-ITPRIPL1 stable transfection cells, and resuspend them in PBS to a cell density of 1.5x10 ⁷ per milliliter. The mice were shaved, and 1.5×10 ⁶ ITPRIPL1-overexpressed MC38 cells were inoculated subcutaneously in the right underarm, which was the construction of an in vivo model of humanized CD3ε mice with MC38ITPRIPL1-overexpressed subcutaneous tumor xenografts. From the fifth day of tumor inoculation, MC38ITPRIPL1-overexpressing xenografted mice in each group were intraperitoneally injected with 100 micrograms of RBD1-binding antibody or the same amount of PBS every three days, and then injected every three days for a total of four treatments. The vernier caliper was used to measure the tumor size, the long diameter and short diameter of the tumor were measured each time, the tumor volume was calculated by the formula of 1/2*A*a*a, and the tumor volume was recorded every three days. On the 23rd day after inoculation, the mice were sacrificed uniformly, and after the tumors were peeled off, the tumor weights were weighed and counted. As shown in Figure 71, both tumor volume and tumor weight showed that tumor growth was significantly inhibited after treatment with RBD1-binding antibody, confirming that ITPRIPL1 monoclonal antibody has function in vivo.

c) Flow cytometry showed that ITPRIPL1 monoclonal antibody can significantly increase the activity of T cells in ITPRIPL1 overexpressing tumors. At least 1 ml of peripheral blood was obtained from the mouse by means of cardiac blood collection. Mouse PBMC cells were obtained by using a mouse peripheral blood PBMC isolation kit (Solarbio, Beijing, China) and operating according to the corresponding instructions of the kit. Mouse PBMCs were placed in EP tubes (Axygen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), and the centrifugation and washing were repeated. Dilute mouse CD8-APC antibody (Biolegend, CA, U.S.), mouse CD69-APC antibody (Biolegend, CA, U.S.), mouse CD137-APC antibody (Biolegend, CA, U.S.) each EP with cell staining buffer at 1:20 Add 200 microliters of the mixture to the tube to resuspend, and incubate at room temperature for 30 minutes with slow shaking. After centrifugation at 400 rcf for 5 minutes, discard the supernatant, resuspend and wash the cells with 500 microliters of cell staining buffer; repeat the centrifugation and washing. Add 200 microliters of cell staining buffer to each, transfer to a flow tube (Falcon, NY, USA) and put it on a machine (Miltenyi, Cologne, Germany). As shown in Figure 72, according to the positive rate of cell fluorescence, the RBD1-binding antibody can significantly increase the expression of CD8, CD25, and CD139. The results of this experiment indicate that RBD1-binding antibodies can relieve the inhibition of ITPRIPL1 on T cell activity in mice.

d) Immunohistochemical staining shows that RBD1-binding antibody can significantly increase immune cell infiltration in ITPRIPL1-overexpressing tumors. The MC38-ITPRIPL1 overexpressing tumor tissue was stripped and sectioned and embedded in paraffin (Bioos, Wuhan, China). The obtained paraffin sections were subjected to dewaxing, hydration, antigen retrieval and other treatments, and then incubated with mouse CD8 antibody (CST, MA, USA) in a wet box overnight. After rewarming on the next day, secondary antibody incubation was carried out according to the reagent instructions. After DAB staining (Solarbio, Beijing, China) and hematoxylin staining (Solarbio, Beijing, China), air-dry against the alcohol concentration gradient and seal the slides. After the slides were sealed, they were observed under a fluorescent microscope using natural light and photographed. As shown in Figure 73, the use of RBD1-binding antibodies can significantly increase the positive rate of CD8 in tumor tissues. The results of this experiment indicate that RBD1-binding antibodies can increase the infiltration of immune cells in tumor tissues overexpressing MC38ITPRIPL1.

The above results show that the antibody that specifically recognizes the extracellular region of ITPRIPL1, namely RBD1, can significantly inhibit the growth of tumor cells in vivo. Since ITPRIPL1 is simultaneously expressed in primary cancer, lymphatic metastatic cancer, and distant metastatic cancer, this allows antibody drugs to act on tumors at different locations and stages of development, and exert more sustained and extensive anti-tumor effects through ADCC, ADCP, and CDC. effect and activate the local immune response in the tumor. At the same time, since the expression of ITPRIPL1 in the control normal tissues of the above tumors is very low, the possible toxic side effects of this antibody against ITPRIPL1 on normal tissues and cells are more limited. The above characteristics highlight the outstanding advantages and application prospects of ITPRIPL1-specific antibodies as a new anti-cancer therapy. Example 7: Regulating the activation of the proliferation signaling pathway of T cell-derived cell lines by regulating the combination of ITPRIPL1-RBD and the extracellular region of CD3

Since previous studies have shown that the combination of CD3ε may cause changes in the proliferation and function of T cells, and because T cells are primary cells that are not conducive to intervention and detection, the most widely used model for such studies is Jurkat cells, which are derived from T cells. cell lines, and the signal transduction of NF-KB is widely used to reflect the detection index of Jurkat or T cell activation degree. Therefore, we constructed the Jurkat-NFKB reporter cell line (the firefly luciferase downstream of the NF-KB promoter was transfected into Jurkat cells through lentivirus, which can be activated by concanavalin A after testing) to detect the expression of ITPRIPL1 in tumor cells The effect on the functional status of co-cultured T cells in the case of

First, the luciferin reporter experiment proved that HCT116 cells overexpressing ITPRIPL1 could more reduce the NFKB proliferation signal of Jurkat-dual cells, as shown in Figure 23 . Count the cultured Jurkat-dual cells, centrifuge at 1000rpm x 5 minutes and resuspend with antibiotic-free IMDM medium to adjust the number of cells to ^2x106 per milliliter, add 200 microliters of cells to each well of a transparent 96-well plate. HCT116 cells and HCT116-ITPRIPL1 stable transfection cells were trypsinized and counted, the cell number was adjusted to 2x10 ⁶ per milliliter, and 20 microliters of each were added to mix with the corresponding Jurkat-dual cells. Placed in a cell culture incubator for co-cultivation for 18-24 hours. After the reaction is completed, take an opaque 96-well plate, add 50 microliters of Quanti-luc reagent and 20 microliters of reaction mixture to each well, and immediately detect the signal with a multi-functional microplate reader after adding and mixing. The figure above reflects that HCT116 cells expressing ITPRIPL1 can inhibit the NFKB proliferation signal of Jurkat-dual cells, while HCT116 cells overexpressing ITPRIPL1 can further reduce the NFKB proliferation signal. The above results indicated that HCT116 cells overexpressing ITPRIPL1 could reduce the NFKB proliferation signal of Jurkat-dual cells more.

Further, the luciferin reporter experiment proved that CD3ε protein can block the inhibition of ITPRIPL1 protein on the NFKB proliferation signal of Jurkat-dual cells, as shown in FIG. 24 . The cultured Jurkat-dual cells were counted, centrifuged at 1000rpm x 5 minutes and then resuspended in IMDM medium without antibiotics to adjust the cell number to ^2x106 per milliliter, and 200 microliters of cells were added to each well of a transparent 96-well plate. 2 micrograms per milliliter of ITPRIPL1 protein and 0 micrograms per milliliter, 1 microgram per milliliter, 2 micrograms per milliliter, and 4 micrograms per milliliter of CD3ε protein mixture were added to each well. After being placed in a cell culture incubator for two hours of reaction, 50 micrograms per milliliter of concanavalin A (ConA) was added to each well, and placed in a cell incubator for 18-24 hours of reaction. After the reaction is completed, take an opaque 96-well plate, add 50 microliters of Quanti-luc reagent and 20 microliters of reaction mixture to each well, and immediately detect the signal with a multi-functional microplate reader after adding and mixing. The above figure reflects the NFKB signal changes of Jurkat-dual cells under the condition of 50 micrograms per milliliter of ConA activation, adding 2 micrograms per milliliter of ITPRIPL1 protein and different concentrations of CD3ε protein. The above results indicated that the inhibition of ITPRIPL1 protein on the NFKB proliferation signal of ConA-activated Jurkat-dual cells could be blocked by CD3ε protein in a concentration-dependent manner, thus indicating that the inhibitory effect of ITPRIPL1 was produced through CD3ε.

Example 8: ITPRIPL1-RBD recombinant protein can reduce the killing of human peripheral blood mononuclear cells (PBMC) to kidney-derived H3K293 cells

Using flow cytometry to demonstrate that ITPRIPL1-RBD recombinant protein reduces the killing of kidney-derived H3K293 cells by human peripheral blood mononuclear cells (PBMCs). CD3 and CD28 antibodies (Invitrogen, CA, USA) were mixed and diluted with PBMCs (ATCC, VA, USA) so that the final concentration was 1 microgram per milliliter to activate T cells, and cultured overnight. The next day, the PBMC and 293E (ATCC, VA, U.S.) cells were counted, and the cell numbers were adjusted to 1× ¹⁰⁶ per milliliter. The control group added 100 microliters of 293E cells and 100 microliters of culture medium, and the experimental group added 100 microliters of each of the two types of cells. Rising to a 96-well plate (Thermo Fisher, MA, U.S.), and adding 1, 2, 4, and 8 micrograms per milliliter of ITPRIPL1-RBD recombinant protein to each of the four groups in the experimental group, and incubated in an incubator for 6 hours . The 96-well plate was taken out, the cells in each well were placed in EP tubes (Axygen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), and the centrifugation and washing were repeated. The CD45-APC antibody (Invitrogen, CA, USA) was diluted 1:20 with cell staining buffer, 200 microliters of the mixture was added to each EP tube to resuspend, and incubated at room temperature for 30 minutes with slow shaking. After centrifugation at 400 rcf for 5 minutes, the supernatant was discarded, and the cells were resuspended and washed with 1 ml of binding buffer (Meilun, Shanghai, China); the centrifugation and washing were repeated. Set without adding PBMC non-staining group, without adding PBMC double-staining group, adding PBMC non-staining group, adding PBMC single-stained Annexin V-FITC group, adding PBMC single-stained PI group, adding PBMC double-stained group and adding ITPRIPL1 protein double To the staining group, add 100 microliters of binding buffer, 5 microliters of Annexin V-FITC (Meilun, Shanghai, China) and 10 microliters of PI (Meilun, Shanghai, China) according to the conditions, incubate at room temperature for 15 minutes with slow shaking, and then Add 400 microliters of binding buffer to each, and transfer to a flow tube (Falcon, NY, USA) and a machine (Miltenyi, Cologne, Germany).

(a) and (b) of FIG. 25 show the division of 293E cells based on CD45. (c) of FIG. 25 is the relative killing activity of PBMC calculated based on the apoptosis data of each group under different ITPRIPL1 protein concentration conditions. Figure 26 shows the specific apoptosis staining conditions under each condition in (c) of Figure 25 . The above results indicate that ITPRIPL1-RBD recombinant protein can reduce the killing of human peripheral blood mononuclear cells (PBMC) to kidney-derived H3K293 cells.

Example 9: Knocking out ITPRIPL1 expressed by tumor cells by gene editing can significantly increase the killing of tumor cells by human peripheral blood mononuclear cells

1. Construction of a CRISPR/Cas9 gene editing system, that is, a lentivirus containing puromycin-resistant sgRNA that specifically cuts ITPRIPL1.

The overall process of the experiment: first synthesize the single-stranded DNA oligo of the gRNA sequence, then anneal and pair to generate a double-stranded DNA oligo, and then directly connect it to the CRISPR/Cas9 vector after digestion through the restriction sites at both ends; The ligation product was transferred into the prepared bacterial competent cells, and the grown monoclonal colony was sent to the sequencing company for sequencing identification, and the correct clone was the successfully constructed CRISPR/Cas9 vector. For the target gene sequence of the target gene, use the gRNA sequence design principles provided in the public website to design multiple target site sequences, as shown in SEQ ID NOs: 11-13. Design and synthesize 3 pairs of gRNA oligomeric single-stranded DNA according to the gene sequence, the oligo sequences are shown in SEQ ID NOs: 14-19, where SEQ ID NO: 14, 15 are gRNA oligomeric corresponding to the target sequence SEQ ID NO: 11 Single-stranded DNA sequence; SEQ ID NO:16,17 is the gRNA oligomeric single-stranded DNA sequence corresponding to the target sequence SEQ ID NO:12; SEQ ID NO:18,19 is the gRNA oligomeric corresponding to the target sequence SEQ ID NO:13 Single-stranded DNA sequence. The oligomeric single-stranded DNA was annealed into double strands, and the double-stranded gRNA oligos were respectively inserted into the CRISPR/Cas9 vectors to construct the CRISPR/Cas9 recombinant plasmids and transformed into competent cells Stbl3. The construction of the carrier includes the following specific steps:

1) Annealing of gRNA: Dilute the primers with sterile TE buffer to make the final concentration 100 μM. Pipette 10 μl of upstream and downstream primers to mix and pipette evenly into the PCR tube for annealing. After completion, place on ice for a few minutes for direct connection or freeze storage at -20°C.

2) Enzyme digestion and recovery of CRISPR/Cas9 vector: The enzyme digestion system is as follows: CRISPR/Cas9vector 5 μg; 10*Buffer 5 μl; BsmBI 4 μl; ddH2O supplement 50 μl. Digest at 37°C for about 30 minutes. During this period, a 0.8% agarose gel can be configured, and nucleic acid electrophoresis can be performed after the enzyme digestion is completed. After electrophoresis, cut out the gel strip containing the fragment of interest. Use a balance to weigh the total weight and subtract the weight of the empty tube to calculate the weight of the gel. Calculate the volume of the gel as 100 mg is about 100 μl, and add QG buffer 3 times the volume of the gel and place it in a 50°C water bath to completely dry the gel. During melting, shake the EP tube properly to speed up the dissolution of the gel. After the gel has completely melted, add an equal volume of isopropanol to the gel and mix well. Transfer all the above liquid to the filter column and centrifuge at 13000rpm for 30s. Discard the liquid in the tube, add 750 μl of PE buffer to the column, and centrifuge for 1 min. Discard the liquid in the tube and centrifuge again for 1 min. Change to a new 1.5ml EP tube, add 50μl EB buffer to the column, centrifuge for 1min, and the recovered carrier is in the centrifuge tube.

3) Connection of CRISPR/Cas9 vector and primers. The ligation system was as follows: recovery vector 3μl (50ng); Oligo primer 1μl (0.5μM); T4DNA ligase buffer 1.5μl; T4DNA ligase 1μl; ddH2O supplement 15μl. Incubate for 30 min in a water bath at 25°C.

4) Transformation: place the competent cells on ice and wait for them to thaw naturally, then add all the ligation products into the competent cells, place them on ice for 20 min, and then heat-shock in a water bath at 42°C for 90 seconds. Then quickly place it on ice for 2-3min. Add 1000 μl of LB without antibiotics and culture based on 37°C, shaking at 150rpm for 45min. Centrifuge at 3000rpm for 2 minutes, discard about 850μl of supernatant, blow and disperse the bacterial solution at the bottom of the tube, add it to the culture dish containing the corresponding resistance, spread it evenly with a sterilized applicator, and place it upside down at 37°C for constant temperature cultivation Incubate overnight in the box.

5) Preparation of recombinant plasmids: Pick several single colonies and carry out a small amount of shaking culture.

6) Positive clones were identified by sequencing. After comparison, the sequence of the insert fragment in the recombinant clone was completely consistent with the designed oligo sequence, so the vector was constructed successfully.

2. Use the CRISPR/Cas9 gene editing system to edit the gene and construct the HCT116-ITPRIPL1 knockout cell line.

Place HCT116 cells (ATCC, VA, U.S.) in a 24-well plate (Corning, NY, U.S.) at an appropriate density (grow to a density of about 30-40% on the second day), and digest and count the next day. The GFP control lentivirus (Jiman, Shanghai, China) was used to infect the cells with a quantitative gradient, the medium was changed after 48 hours, and GFP fluorescence was observed under the microscope after 72 hours to determine the optimal virus-to-cell infection ratio and explore the MOI value. After determining the MOI value, re-plate, infect HCT116 cells with the Cas9 system lentivirus containing blasticidin resistance (Jiman, Shanghai, China) at the MOI value, change the medium after 48 hours, and add concentration gradient after 72 hours Blasticidin (Invivogen, CA, USA) was used for screening for 10-14 days, and the cell line obtained from the final screening was HCT116 cells containing the Cas9 system. Then, HCT116 cells containing the Cas9 system were plated and counted in a 24-well plate, and infected with a lentivirus containing puromycin-resistant sgRNA that specifically cuts ITPRIPL1 at an MOI value, and the medium was changed after 48 hours, and the concentration gradient was entered after 72 hours Puromycin (Invivogen, CA, USA) was selected for 10-14 days to obtain the HCT116-ITPRIPL1 knockout cell line.

3. Flow cytometry proved that knocking down ITPRIPL1 expressed by tumor cells can significantly increase the killing of human peripheral blood mononuclear cells to tumor cells.

CD3 and CD28 antibodies (Invitrogen, CA, USA) were mixed and diluted with PBMCs (ATCC, VA, USA) so that the final concentration was 1 microgram per milliliter to activate T cells, and cultured overnight. The next day, PBMC and HCT116 wild-type (ATCC, VA, USA)/ITPRIPL1 overexpression/ITPRIPL1 knockout cells were counted, and the number of cells was adjusted to ^1x106 per milliliter, and 100 microliters of tumor cells and 100 microliters of culture medium were added to the control group 100 microliters of tumor cells and PBMCs in the experimental group were added to a 96-well plate (Thermo Fisher, MA, USA), and incubated in an incubator for 6 hours. Take out the 96-well plate, rinse with PBS (Meilun, Dalian, China), digest the cells with EDTA-free trypsin (Beyond, Shanghai, China), and place in EP tubes (Axygen, CA, USA), 400 rcf Discard the supernatant after centrifugation for 5 minutes. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), and the centrifugation and washing were repeated. The CD45-APC antibody (Invitrogen, CA, USA) was diluted 1:20 with cell staining buffer, 200 microliters of the mixture was added to each EP tube to resuspend, and incubated at room temperature for 30 minutes with slow shaking. After centrifugation at 400 rcf for 5 minutes, the supernatant was discarded, and the cells were resuspended and washed with 1 ml of binding buffer (Meilun, Dalian, China); the centrifugation and washing were repeated. Set without adding PBMC non-staining group, without adding PBMC double-staining group, adding PBMC non-staining group, adding PBMC single-stained Annexin V-FITC group, adding PBMC single-stained PI group, adding PBMC double-stained group and each double-stained group with IT1 protein To the staining group, add 100 microliters of binding buffer, 5 microliters of Annexin V-FITC (Meilun, Dalian, China) and 10 microliters of PI (Meilun, Dalian, China) according to the conditions, and incubate with slow shaking at room temperature for 15 minutes, then Add 400 microliters of binding buffer to each, and transfer to a flow tube (Falcon, NY, USA) and a machine (Miltenyi, Cologne, Germany).

(a) and (b) of FIG. 27 show that tumor cells are classified according to CD45. (c) of FIG. 27 is the relative killing activity of PBMC calculated based on the apoptosis data of each group under different ITPRIPL1 expression conditions. Figure 28 shows the specific apoptosis staining conditions under each condition in (c) of Figure 27. The above results indicate that overexpression of ITPRIPL1 can reduce the killing of PBMCs against tumor cells and knocking out ITPRIPL1 can promote the killing of PBMCs against tumor cells.

Example 10: Antibodies prepared using ITPRIPL1-RBD protein as an immunogen can effectively promote the killing of tumor cells by immune cells

CD3 and CD28 antibodies (Invitrogen, CA, USA) were mixed and diluted with PBMCs (ATCC, VA, USA) so that the final concentration was 1 microgram per milliliter to activate T cells, and cultured overnight. PBMC and HCT116 cells (ATCC, VA, U.S.) were counted the next day, and the cell numbers were adjusted to 1×10 ⁶ per milliliter. Added 100 microliters of HCT116 cells and 100 microliters of culture medium to the control group, and added 100 microliters of each of the two types of cells to the experimental group. Upgrade to a 96-well plate (Thermo Fisher, MA, USA), add 1:500/250/125/62.5 ITPRIPL1 polyclonal antibody to each of the 4 groups in the experimental group, and incubate in an incubator for 6 hours. Take out the 96-well plate, rinse with PBS (Meilun, Shanghai, China) and digest the cells with EDTA-free trypsin (Beyond, Shanghai, China) and place in EP tubes (Axygen, CA, USA), 400 rcf Discard the supernatant after centrifugation for 5 minutes. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), and the centrifugation and washing were repeated. The CD45-APC antibody (Invitrogen, CA, USA) was diluted 1:20 with cell staining buffer, 200 microliters of the mixture was added to each EP tube to resuspend, and incubated at room temperature for 30 minutes with slow shaking. After centrifugation at 400 rcf for 5 minutes, the supernatant was discarded, and the cells were resuspended and washed with 1 ml of binding buffer (Meilun, Shanghai, China); the centrifugation and washing were repeated. Set without adding PBMC non-staining group, without adding PBMC double-staining group, adding PBMC non-staining group, adding PBMC single-stained Annexin V-FITC group, adding PBMC single-stained PI group, adding PBMC double-stained group and adding ITPRIPL1 protein double To the staining group, add 100 microliters of binding buffer, 5 microliters of Annexin V-FITC (Meilun, Shanghai, China) and 10 microliters of PI (Meilun, Shanghai, China) according to the conditions, incubate at room temperature for 15 minutes with slow shaking, and then Add 400 microliters of binding buffer to each, and transfer to a flow tube (Falcon, NY, USA) and a machine (Miltenyi, Cologne, Germany).

(a) and (b) of FIG. 29 show that HCT116 cells were divided according to CD45. (c) of FIG. 29 is the relative killing activity of PBMC calculated based on the apoptosis data of each group under different polyclonal antibody concentration conditions. Figure 30 shows the specific apoptosis staining conditions under each condition in (c) of Figure 29. The above results indicate that the ITPRIPL1 polyclonal antibody can promote the killing of tumor cells by PBMC.

Example 11: The binding ability of ITPRIPL1-RBD2 sequence mutants to CD3E is reduced to a certain extent, and the binding ability of ITPRIPL1-RBD3 to CD3ε is significantly reduced

1. Construction of HCT116-RBD2 and RBD3 sequence mutants

Based on the published sequence (NCBI reference sequence NM_001008949.3), DRMDLDTLARSRQLEKRMSEEMRLLEMEFEERKRAAEQRQKAENFWTGDTSSDQ (ITPRIPL1-RBD2, ie SEQ ID NO: 2), MDLDTLARSRQLEKRMSEEMRLLEMEFEERKRAAEQRQ in the extracellular region (25-103 amino acids) were selected respectively KAEN (ITPRIPL1-RBD3, ie SEQ ID NO :3) Synthesize specific ITPRIPL1 sequence mutants for two different target sequences, using pcDNA3.1 as the vector, in which the extracellular region, the C-terminal of the extracellular region and the transmembrane region are fused to the Flag tag and green fluorescent protein (GFP) , thereby generating ITPRIPL1 extracellular region, ITPRIPL1-RBD2, ITPRIPL1-RBD3 plasmids containing Flag tag and green fluorescent protein (GFP).

2. Co-immunoprecipitation assay ITPRIPL1-RBD2 is the shortest sequence combined with CD3E.

The pcDNA3.1 plasmids containing Flag-tagged ITPRIPL1 extracellular region, ITPRIPL1-RBD2, ITPRIPL1-RBD3 and HA-tagged CD3E were co-transfected into HCT116 cells (ATCC, VA, USA), placed in six-well plates (Corning , NY, U.S.) for 48-72 hours until the protein was fully expressed, the 1:100 immunoprecipitation lysate (Thermo Fisher, MA, U.S.) and protease inhibitor, phosphatase inhibitor, PMSF triple (Conson, Shanghai, China) mixed lysate to lyse and scrape the cells, a part of the cell samples were centrifuged, mixed with loading buffer (Beyond, Shanghai, China) and denatured in a metal bath at 100 degrees Celsius to prepare the input protein samples; the rest of the cell samples were labeled with HA Specific mouse antibody (Biolegend, CA, USA) or Flag tag-specific mouse antibody (CST, MA, USA) was used for immunoprecipitation, washed with PBS, mixed with loading buffer (Biolegend, Shanghai, China) and heated at 100 degrees Celsius Immunoprecipitated protein samples were prepared after bath denaturation. Then configure 12.5% PAGE gel (Yase, Shanghai, China) in the gel plate (Bole, CA, USA) according to the instructions, place the prepared gel in the electrophoresis tank (Bole, CA, USA), and connect the power supply (Bole, CA, U.S.) Let the strips run through the stacking gel with a constant voltage of 80 volts, and run through the separating gel with a constant voltage of 120 volts. When the strips reached the bottom of the separating gel, transfer to the membrane using a wet method in a transfer tank (Bio-Rad, CA, USA) with a constant current of 350 mA for 90 minutes. After the membrane transfer was completed, the membrane was sheared according to the mass size of ITPRIPL1 extracellular region, ITPRIPL1-RBD2, ITPRIPL1-RBD3 and CD3E-HA protein. Block with fast blocking solution (Yase, Shanghai, China) for ten minutes, and incubate the extracellular region of ITPRIPL1 with Flag-tag-specific rabbit antibody (Abcam, MA, USA) and HA-tag-specific rabbit antibody (CST, MA, USA) , ITPRIPL1-RBD2, ITPRIPL1-RBD3 and the corresponding bands of CD3E-HA, four overnight. After washing with TBST the next day, incubate with specific anti-rabbit secondary antibody (Consun, Shanghai, China) diluted in 5% skimmed milk powder (Sangon, Shanghai, China) dissolved in TBS for one hour at room temperature, wash with TBST, and place Exposure to a gel imager (Bio-Rad, CA, USA) for one minute in a mixed luminescence solution (San Er, Shanghai, China).

(a) of Figure 31 shows the content of different ITPRIPL1 sequence mutants and CD3ε in the input protein, and (b) of Figure 31 shows the mutants of different sequences of ITPRIPL1 that are precipitated indirectly (because they are combined with CD3ε), and the mutants of different sequences of ITPRIPL1 that are precipitated directly. CD3ε. (c) of Figure 31 shows the content of different ITPRIPL1 sequence mutants and CD3ε in the input protein, and (d) of Figure 31 shows the CD3E of indirect precipitation (due to the combination of mutants with different sequences of ITPRIPL1), and the content of CD3E of direct precipitation. Mutants with different sequences of ITPRIPL1. The above results indicated that in the presence of the extracellular region of ITPRIPL1 and RBD2 sequence mutants, CD3E and ITPRIPL1 remained bound; while RBD3 sequence could not detect significant binding. From the above, it can be concluded that there is a positive correlation between the length or integrity of the extracellular region of ITPRIPL1 and the ability to bind to CD3E.

Example 12: The isolated ITPRIPL1-RBD protein has the ability to bind NRP2

1. Enzyme-linked immunosorbent assay (ELISA) confirmed the concentration-dependent direct binding of the isolated ITPRIPL1 (IT1) extracellular domain fragment to the extracellular domain of NRP2.

A special plate for ELISA (costar, ME, USA) was used. First, 0.03125/0.0625/0.125/0.25/0.5/1/2 micrograms per milliliter of ITPRIPL1-RBD recombinant protein was dissolved in 100 microliters of ELISA coating solution (Solarbio, Beijing, China) to coat the plate, and the negative control was protein-free 100 µl of coating solution to coat the plate. Four times wrapped overnight. After washing with PBST, 100 microliters of 5% BSA dissolved in PBS (VWR, PA, USA) was used to block, and the incubator was blocked at 37 degrees for 90 minutes. After washing with PBST, incubate and bind with 1 microgram per milliliter of NRP2 extracellular region protein fragment (Shenzhou, Beijing, China). minute. After washing with PBST, specific anti-human Fc fragment antibody (Abcam, MA, USA) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, use chromogenic solution (Sangon, Shanghai, China) for color development, 100 microliters per well, place in an incubator for 5-30 minutes, and then add 50 microliters of stop solution (Sangon, Shanghai, China) ), placed in a microplate reader (Thermo Fisher, MA, USA) for chromogenic reading at 450 nm.

As shown in Figure 32, this experiment demonstrated concentration-dependent direct binding of the isolated ITPRIPL1 extracellular domain fragment to the NRP2 extracellular domain. The above results prove that the isolated purified extracellular domain protein of ITPRIPL1 (IT1-RBD) binds directly to the purified extracellular domain protein of NRP2 in a concentration-dependent manner.

2. Flow cytometry proves that NRP2 binds to cells overexpressing ITPRIPL1 with higher efficiency.

After detection, HEK293 cells endogenously express a certain level of ITPRIPL1, and the expression level further increases after stable expression. The cultured 293E wild-type cells (ATCC, VA, U.S.) and 293E-ITPRIPL1 stable transfection cells were digested and counted, and the number of cells was adjusted to 2× ¹⁰⁶ per milliliter, and 200 microliters were added to 96-well plates (costar, ME, USA) in each well. 0/0.5/1/2/4 micrograms per milliliter of NRP2 protein (Shenzhou, Beijing, China) were added to the wells of each experimental group. The 96-well plate was placed in a cell culture incubator and allowed to stand for 30 minutes, then removed, transferred to an EP tube (Axygen, CA, USA) and centrifuged at 400 rcf for 5 minutes, then the supernatant was discarded. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded again and the washing was repeated. Dilute anti-human IgG Alexa Fluor 647 antibody (Invitrogen, CA, USA) 1:1000 with cell staining buffer, add 200 microliters of antibody diluent to each EP tube except the negative control, and incubate at room temperature on a shaker at 40 rpm for 30 minute. Then take out the EP tube, centrifuge at 400 rcf for 5 minutes and discard the supernatant. Wash the cells by resuspending them with 1000 μl of cell staining buffer, centrifuge at 400 rcf for 5 minutes, discard the supernatant again and repeat the washing. After washing, each EP tube was resuspended by adding 300 microliters of cell staining buffer, and transferred to a flow tube (Falcon, NY, USA) for on-machine analysis (Miltenyi, Cologne, Germany).

(a) of FIG. 33 is the threshold setting for not classifying dead cells. (b) of FIG. 33 reflects the binding status of cells with different expression status of ITPRIPL1 to NRP2 protein. Figure 34 shows the specific staining and protein binding conditions under each condition in (b) of Figure 33 . The above results indicate that NRP2 can bind to 293E cells with a certain expression of ITPRIPL1, and can bind more strongly to 293E cells overexpressing ITPRIPL1.

Example 13: The isolated ITPRIPL1-RBD protein has the ability to transmit inhibitory signals to differentiated THP1 macrophages expressing NRP2 protein

The luciferin reporter experiment proved that the free isolated ITPRIPL1 extracellular domain purified protein (IT1-RBD) could inhibit the IFN proliferation signal of THP1-dual cells induced by PMA under inactive conditions.

The cultured THP1-dual cells (Invivogen, CA, the U.S.) were counted, centrifuged at 1000 rpm for 5 minutes, and then resuspended with antibiotic-free RPMI-1640 medium (Gibco, CA, the U.S.) to adjust the number of cells to ^2x10 per milliliter, transparent 96 Add 200 microliters of cells to each well of a well plate (Thermo Fisher, MA, USA) and induce with 20 ng/ml of PMA reagent (Invivogen, CA, USA). After culturing in the incubator for 3 hours, wash with PBS and replace the medium. After 72 hours, the medium was changed again and 0/1/2/4/8 micrograms per milliliter of purified protein from the extracellular region of free isolated ITPRIPL1 were added, and cultured in a cell culture incubator for 18-24 hours. After the reaction was completed, an opaque 96-well plate was taken, and 50 microliters of Quanti-luc reagent (Invivogen, CA, USA) and 10 microliters of cell mixture were added to each well, and the signal was detected immediately with a multifunctional microplate reader after adding and mixing.

As shown in Figure 35, the above results indicated that the free isolated purified extracellular region of ITPRIPL1 protein (IT1-RBD) could inhibit the IFN proliferation signal of THP1-dual cells induced by PMA under inactive conditions. The ITPRIPL1-RBD protein isolated from above has the ability to deliver inhibitory signals to differentiated THP1 macrophages expressing NRP2 protein.

Example 14: The purified IT1-RBD-Fc protein has the function of inhibiting T cell signaling and killing

1. Separation and purification of IT1-RBD-Fc protein.

Based on the published sequence (NCBI reference sequence NM_001008949.3), the extracellular region (HPLMVSDRMDLDTLARSRQLEKRMSEEMRLLEMEFEERKRAAEQRQKAENFWTGDTSSDQLVLGKKDMGWPFQADGQEG) was selected as IT1-RBD1, that is, the extracellular region removed all parts of the signal peptide; DRMDLDTLARSRQLEKRMSEEMRLLEME FEERKRAAEQRQKAENFWTGDTSSDQ as IT1-RBD2, that is, go on the basis of RBD1 In addition to multiple conserved sequence amino acids at the N-terminus, as well as multiple conserved and non-conserved sequence amino acids at the C-terminus (used to verify the importance of mammalian conserved amino acid positions for function);

MDLDTLARSRQLEKRMSEEMRLLEMEFEERKRAAEQRQKAEN as IT1-RBD3 (only the sequence predicted to be the secondary structure of Alpha helix is retained, and it is verified whether the secondary structure is the minimum sequence required for function), followed by tandem Fc sequence without CH1 region

(PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK), using pcDNA3.1 as the vector to construct the corresponding plasmid. The above-mentioned mutation-derived sequences of several extracellular regions of ITPRIPL1 are shown in FIG. 54 .

Through this example, the functional sequence of the extracellular region of ITPRIPL1 was characterized. Using the AlphaFold predicted structure of the functional fragment of the extracellular region of ITPRIPL1 and the X-ray diffraction crystal structure of CD3E (1XIW.pdb) for protein-protein docking, the analysis shows the human, mouse, rat, green monkey, and golden monkey shown in Figure 54 ITPRIPL1 of various species such as , black snub-nosed monkey, Bolivian squirrel monkey, Martensian nocturnal monkey, and chimpanzee can bind to CD3E, which is consistent with the results of immunofluorescence and colocalization analysis in Example 2.

Therefore, according to the differential sites of different species, the RBD1-derived sequence with the function of binding CD3E is obtained:

DRMDLDTLARSRQLEKRMSEEMRxLEMEFEERxxxAExxQKxENxWxGxTSxDQ (where x represents an alternate amino acid).

In this embodiment, functional detection and analysis were performed on RBD1, RBD2, and RBD3.

After obtaining the plasmid, it was transfected into 293E cells (ATCC, VA, USA) by PEI reagent (Liji, Shanghai, China). Enzyme inhibitor, PMSF triple (Consun, Shanghai, China) lysed cells on ice for 10 minutes, centrifuged at 12,000rpm for 15 minutes, took out the supernatant, and used 1:100 protein A magnetic beads (Tiandirenhe, Changzhou, China) to shake at room temperature at 130rpm For 2 hours, the mixed solution was sucked into a filter column (Millipore, MA, U.S.) equilibrated with a balance solution (20mM disodium hydrogen phosphate, 0.15M sodium chloride, pH7.0) in advance, washed twice with the balance solution, and washed with Eluting solution (0.1M glycine, pH3.0): neutralizing solution (1M Tris-HCl, pH8.5) = 16:1 for elution, and the eluted product is the purified IT1-RBD-Fc protein. The protein concentration was measured with a Nanodrop spectrophotometer and stored at -20°C.

2. The luciferin reporter experiment proved that the purified IT1-RBD1-Fc protein can inhibit the NFKB proliferation signal of Jurkat-dual cells under ConA activation and inactivation conditions.

Cultured Jurkat-dual cells (Invivogen, CA, U.S.) were counted, centrifuged at 1000rpmx for 5 minutes and then resuspended in antibiotic-free IMDM medium (Gibco, CA, U.S.) to adjust the number of cells to ^2x10 per milliliter, in a transparent 96-well plate Add 200 μl of cells per well. 0/4 microgram per milliliter of purified IT1-RBD1/RBD2/RBD3 Fc protein was added to each well. After two hours of reaction in the cell culture incubator, 50 micrograms per milliliter of concanavalin A (ConA) (Aladdin, Shanghai, China) was added to each well as the activation group or an equal volume of endotoxin-free water as the non-activation group. React in the cell culture incubator for 18-24 hours. After the reaction is completed, take an opaque 96-well plate (costar, ME, USA), add 50 microliters of Quanti-luc reagent (Invivogen, CA, USA) and 20 microliters of reaction mixture to each well, add and mix well, and then use the multifunctional enzyme The marker immediately detects the signal.

As shown in Figure 36, the NFKB signal changes in Jurkat-dual cells under the condition of 50 micrograms per milliliter of ConA activation and inactivation, and the addition of 4 micrograms per milliliter of Fc proteins of different IT1-RBD segments. The above results indicated that the purified IT1-RBD1-Fc protein could inhibit the NFKB proliferation signal of Jurkat-dual cells under ConA activation and inactivation conditions, and the inhibitory effect gradually weakened as the sequence gradually shortened.

3. Flow cytometry proved that the purified IT1-RBD1-Fc recombinant protein can reduce the killing of human peripheral blood mononuclear cells (PBMC) to kidney-derived H3K293 cells.

HEK293 cells are widely used to create autoimmune disease models, conduct renal autoimmune diseases, nervous system autoimmune diseases, systemic lupus erythematosus (SLE), etc., and conduct research on disease progression, molecular cell biology mechanisms, and pharmacology (Stepanenko AA , Gene.2015Sep 15;569(2):182-90, Hira S.J Physiol Sci.2019Sep;69(5):723-732., Keskitalo S,, Front Immunol.2019Dec 5;10:2770.). In addition, HEK293 cells have also been used to construct models of rejection disease after transplantation (Yi Gao.Int J Clin Exp Med.2014; 7(11):4572–4583, Lorber, Marc I, Transplantation:1999–67(6)-p 897-903, Pabois A, Biochem Pharmacol.2016Mar 15;104:95-107), antiviral infection immune response (Ismail Cem Yilmaz, Allergy.2021Sep 14.doi:10.1111/all.15091, Elizabeth A Reap,Vaccine.2007Oct 16;25(42):7441-9), tumor immune response, etc. (A A Stepanenko, Gene. 2015Sep 15;569(2):182-90).

In addition, peripheral blood mononuclear cells (PBMC) derived from autoimmune diseases or normal people are also used in many studies to construct autoimmune disease models (Yoshikawa N. Horm Metab Res.1994Sep; 26(9):419-23. ), post-transplant rejection (Transplant Proc.2016Oct; 48(8):2840-2844.), anti-infection immune response (Har-Noy M, J Transl Med.2020May 12; 18(1):196, Nakamura-Hoshi M , Sci Rep.2020Jul 9; 10(1):11394.), anti-tumor immune response, etc. (Zhuang X, Cancer Immunol Res.2019Jun; 7(6):939-951), the target cells involved also include HEK293 cell. In fact, the effect of the NSG mouse model based on PBMC humanization is highly related to the interaction between PBMC and target cells in vitro. Therefore, the in vitro model based on PBMC and target cells has a role that cannot be ignored in the manifestation of disease process and drug efficacy. .

In this example, HEK293 cells are used as target cells for autoimmunity, transplant rejection, allergic reactions, and anti-tumor reactions, and human peripheral blood mononuclear cells (PBMC) are used as effector cells. Through representative disease models, the regulation based on ITPRIPL1 is demonstrated. The effect of agents on the interaction between antigen presenting cells and T cells, as well as the intervention of the above-mentioned different diseases.

The implementation process is described in detail as follows: CD3 and CD28 antibodies (Invitrogen, CA, USA) were mixed and diluted with PBMCs (ATCC, VA, USA) so that the final concentration was 1 microgram per milliliter to activate T cells, and cultured overnight. The next day, the PBMC and 293E wild-type (ATCC, VA, U.S.)/ITPRIPL1 overexpression cells were counted, and the cell number was adjusted to 1× ¹⁰⁶ per milliliter, and the control group was added with 100 microliters of 293E wild-type/ITPRIPL1 overexpression cells and 100 microliters In the experimental group, 100 microliters of PBMC and another cell were added to a 96-well plate (costar, ME, USA). In the experimental group, 2 micrograms per milliliter of IT1- RBD1/RBD2/RBD3-Fc protein was incubated in an incubator for 6 hours. The 96-well plate was taken out, the cells were absorbed, placed in EP tubes (Axygen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), and the centrifugation and washing were repeated. The CD45-APC antibody (Invitrogen, CA, USA) was diluted 1:20 with cell staining buffer, 200 microliters of the mixture was added to each EP tube to resuspend, and incubated at room temperature for 30 minutes with slow shaking. After centrifugation at 400 rcf for 5 minutes, the supernatant was discarded, and the cells were resuspended and washed with 1 ml of binding buffer (Beyond, Shanghai, China); the centrifugation and washing were repeated. Set without adding PBMC non-staining group, without adding PBMC double-staining group, adding PBMC non-staining group, adding PBMC single-stained Annexin V-FITC group, adding PBMC single-stained PI group, adding PBMC double-stained group and each double-stained group with IT1 protein To the staining group, add 100 microliters of binding buffer, 5 microliters of Annexin V-FITC (Beyotime, Shanghai, China) and 10 microliters of PI (Beyotime, Shanghai, China) according to the conditions, and incubate with slow shaking at room temperature for 15 minutes, and then Add 400 microliters of binding buffer to each, and transfer to a flow tube (Falcon, NY, USA) and a machine (Miltenyi, Cologne, Germany).

(a) of FIG. 37 shows the division of 293E cells according to CD45. (b) of FIG. 37 is the relative killing activity of PBMC calculated based on the apoptosis data of each group under different ITPRIPL1 expression and different protein conditions. Figure 38 shows the specific apoptosis staining conditions under each condition in (b) of Figure 37. The above results show that the purified IT1-RBD1-Fc recombinant protein can reduce the killing effect of human peripheral blood mononuclear cells (PBMC) on kidney-derived H3K293 cells, and the reduction effect gradually weakens as the sequence gradually shortens.

4. Western blot experiments showed that the ITPRIPL1-RBD recombinant protein and the purified IT1-RBD1-Fc recombinant protein had the function of inhibiting the ZAP70 and Akt pathways downstream of CD3E.

The cultured Jurkat cells (ATCC, VA, the U.S.) were counted, and 4× ¹⁰ cells were transferred to a 15ml centrifuge tube (Corning, NY, the U.S.), discarded after centrifugation at 800 rpm for 4 minutes, and replaced with serum-free RPMI1640 medium (U.S. Lun, Dalian, China) was resuspended, placed in an incubator for starvation culture for 3 hours, centrifuged at 800 rpm for 4 minutes, 4 ml of complete medium (Meilun, Dalian, China) was resuspended, and 1 ml each was placed in a 12-well plate (Corning, NY, USA) to each well, each well was added: no treatment; 4 micrograms per milliliter of IT1-RBD protein; 4 micrograms per milliliter of RBD1-Fc protein; 4 micrograms per milliliter of RBD2-Fc protein. Mix well, place in the incubator for 10min and take it out, transfer the cells to a 1.5ml EP tube (Axygen, CA, USA), centrifuge at 800rpm for 4 minutes, resuspend and wash with PBS, and repeat the washing. After the centrifugation was completed, the supernatant was discarded, and RIPA lysate (Beyond, Shanghai, China) was mixed with protease inhibitor, phosphatase inhibitor, and PMSF (Consun, Shanghai, China) at a ratio of 1:100, and 80 microliters of cells were added to each tube. liter of mixed lysate. Each EP tube was frozen three times and thawed three times on liquid nitrogen-ice, and centrifuged at 12000 rpm at 4 degrees Celsius for 15 minutes after the last thaw. After the centrifugation, take the supernatant. After the centrifugation, the supernatant: 5x sample buffer (Beyond, Shanghai, China) is prepared at a ratio of 4:1 to each cell sample, and placed in a metal bath at 100 degrees Celsius for 10 minutes to denature to prepare a protein sample. . Then configure 10% PAGE gel (Yase, Shanghai, China) in the gel plate (Bole, CA, USA) according to the instructions, put the prepared gel in the electrophoresis tank (Bole, CA, USA), and connect the power supply (Bole, CA, U.S.) Let the strips run through the stacking gel with a constant voltage of 80 volts, and run through the separating gel with a constant voltage of 120 volts. When the strips reached the bottom of the separating gel, transfer to the membrane using a wet method in a transfer tank (Bio-Rad, CA, USA) with a constant current of 350 mA for 90 minutes. After the membrane transfer was completed, the membrane was sheared according to the mass of Akt, ZAP70 and GAPDH proteins. Block with rapid blocking solution (Yase, Shanghai, China) for ten minutes, and use pAkt-specific rabbit antibody (CST, MA, USA), Akt-specific rabbit antibody (CST, MA, USA), pZAP70-specific rabbit antibody (CST , MA, USA), GAPDH antibody (Consun, Shanghai, China) were incubated with the corresponding bands, overnight four times. After washing with TBST the next day, incubate with specific anti-rabbit secondary antibody (Consun, Shanghai, China) diluted in 5% skimmed milk powder (Sangon, Shanghai, China) dissolved in TBS for one hour at room temperature, wash with TBST, and place Exposure to a gel imager (Bio-Rad, CA, USA) for one minute in a mixed luminescence solution (San Er, Shanghai, China).

Figure 39 (a) and (b) respectively show the expression of phosphorylated Akt and phosphorylated ZAP70 after adjusting the GAPDH internal reference in Western blotting, where Akt phosphorylation and ZAP70 phosphorylation were obtained by ITPRIPL1-RBD recombinant protein and purification After treatment, the IT1-RBD-Fc protein decreased to a certain extent, and the phosphorylation of the IT1-RBD1-Fc recombinant protein decreased more significantly than that of the IT1-RBD2-Fc protein. The experimental results showed that the ITPRIPL1-RBD recombinant protein and the purified IT1-RBD1-Fc protein had the effect of reducing the phosphorylation of Akt and ZAP70 downstream of CD3E, thus indicating that they had the function of inhibiting the corresponding T cell pathway.

5. Western blot experiments showed that CD3E protein has the function of blocking the effect of ITPRIPL1-RBD1-Fc recombinant protein on phosphorylated ZAP70 and Akt pathways.

The cultured Jurkat cells (ATCC, VA, the U.S.) were counted, and 4× ¹⁰ cells were transferred to a 15ml centrifuge tube (Corning, NY, the U.S.), discarded after centrifugation at 800 rpm for 4 minutes, and replaced with serum-free RPMI1640 medium (U.S. Lun, Dalian, China) was resuspended, placed in an incubator for starvation culture for 3 hours, centrifuged at 800 rpm for 4 minutes, 4 ml of complete medium (Meilun, Dalian, China) was resuspended, and 1 ml each was placed in a 12-well plate (Corning, NY, USA) to each well, each well was added: no treatment; 4 micrograms per milliliter RBD1-Fc protein; 4 micrograms per milliliter RBD1-Fc protein + 4 micrograms per milliliter CD3E protein. Mix well, place in the incubator for 10min and take it out, transfer the cells to a 1.5ml EP tube (Axygen, CA, USA), centrifuge at 800rpm for 4 minutes, resuspend and wash with PBS, and repeat the washing. After the centrifugation was completed, the supernatant was discarded, and RIPA lysate (Beyond, Shanghai, China) was mixed with protease inhibitor, phosphatase inhibitor, and PMSF (Consun, Shanghai, China) at a ratio of 1:100, and 80 microliters of cells were added to each tube. liter of mixed lysate. Each EP tube was frozen three times and thawed three times on liquid nitrogen-ice, and centrifuged at 12000 rpm at 4 degrees Celsius for 15 minutes after the last thaw. After the centrifugation, take the supernatant. After the centrifugation, the supernatant: 5x sample buffer (Beyond, Shanghai, China) is prepared at a ratio of 4:1 to each cell sample, and placed in a metal bath at 100 degrees Celsius for 10 minutes to denature to prepare a protein sample. . Then configure 10% PAGE gel (Yase, Shanghai, China) in the gel plate (Bole, CA, USA) according to the instructions, put the prepared gel in the electrophoresis tank (Bole, CA, USA), and connect the power supply (Bole, CA, U.S.) Let the strips run through the stacking gel with a constant voltage of 80 volts, and run through the separating gel with a constant voltage of 120 volts. When the strips reached the bottom of the separating gel, transfer to the membrane using a wet method in a transfer tank (Bio-Rad, CA, USA) with a constant current of 350 mA for 90 minutes. After the membrane transfer was completed, the membrane was sheared according to the mass of Akt, ZAP70, ERK and GAPDH proteins. Block with rapid blocking solution (Yase, Shanghai, China) for ten minutes, and use pAkt-specific rabbit antibody (CST, MA, USA), Akt-specific rabbit antibody (CST, MA, USA), pZAP70-specific rabbit antibody (CST , MA, USA), ZAP70-specific map antibody (CST, MA, USA), pERK-specific rabbit antibody (CST, MA, USA), ERK-specific rabbit antibody (CST, MA, USA), GAPDH antibody (Conson, Shanghai, China) to incubate the corresponding bands separately, four times overnight. After washing with TBST the next day, incubate with specific anti-rabbit secondary antibody (Consun, Shanghai, China) diluted in 5% skimmed milk powder (Sangon, Shanghai, China) dissolved in TBS for one hour at room temperature, wash with TBST, and place Exposure to a gel imager (Bio-Rad, CA, USA) for one minute in a mixed luminescence solution (San Er, Shanghai, China).

Fig. 41 is the result of immunoblotting experiment corresponding to the change of phosphorylation pathway. As shown in the figure, the phosphorylation of Akt, ZAP70, and ERK all decreased significantly after the addition of ITPRIPL1-RBD1-Fc, and ZAP70 was the most obvious; after the addition of CD3E protein, the decline in phosphorylation was offset. The experimental results show that CD3E can block the regulatory effect of ITPRIPL1-RBD1-Fc protein on the phosphorylation pathway, and ITPRIPL1-RBD-Fc regulates the phosphorylation pathway of T cells through CD3.

Example 15: The regulation of T cell function by ITPRIPL1 requires the correct conformational change of CD3 and the PRS segment

1. Construction of CD3 mutant Jurkat cells

The cultured Jurkat cells (ATCC, VA, USA) were counted, and 2×10 ⁵ cells were transferred to each well of a 24-well plate (Corning, NY, USA), and plated overnight. 500 microliters of complete medium (Meilun, Dalian, China) was mixed with CD3 knockout lentivirus (Jima, Shanghai, China) at an MOI value of 100 for virus infection. Change the medium after 48 hours. After culturing for 24 hours, 1 microgram per milliliter of puromycin was added for a week-long selection. After the screening, the CD3 knockout Jurkat cell line was obtained.

The CD3 knockout Jurkat cells were counted, and ^2x105 cells were transferred to each well of a 24-well plate, and plated overnight. Mix 500 microliters of complete medium with CD3-K76T (unable to undergo correct conformational change) and CD3-ΔPRS (PRS segment change) overexpression virus calculated at an MOI of 100 for virus infection. Change the medium after 48 hours. After continuing to culture for 24 hours, 600 micrograms per milliliter of Geneticin (G418) (Gibco, CA, USA) was added for a week-long selection. After the screening, the Jurkat cell line with CD3 mutation was obtained.

2. Western blotting experiments showed that the regulation of ITPRIPL1's phosphorylation pathway on T cells requires the conformational change of CD3 and the PRS segment

Count the wild-type Jurkat cells, CD3 knockout Jurkat cells, CD3-K76T Jurkat cells, and CD3-ΔPRS cells, take ^1x106 cells to each well of a 12-well plate (Corning, NY, USA), add 10 micrograms per milliliter of OKT3 After activation, each was not treated or treated with 4 micrograms per milliliter of ITPRIPL1-RBD1-Fc, and cells were harvested after incubation for 10 minutes. The cells were transferred to a 1.5ml EP tube (Axygen, CA, USA), centrifuged at 800 rpm for 4 minutes, resuspended in PBS, washed and centrifuged, and washed repeatedly. After the centrifugation was completed, the supernatant was discarded, and RIPA lysate (Beyond, Shanghai, China) was mixed with protease inhibitor, phosphatase inhibitor, and PMSF (Consun, Shanghai, China) at a ratio of 1:100, and 80 microliters of cells were added to each tube. liter of mixed lysate. Each EP tube was frozen three times and thawed three times on liquid nitrogen-ice, and centrifuged at 12000 rpm at 4 degrees Celsius for 15 minutes after the last thaw. After the centrifugation, take the supernatant. After the centrifugation, the supernatant: 5x sample buffer (Beyond, Shanghai, China) is prepared at a ratio of 4:1 to each cell sample, and placed in a metal bath at 100 degrees Celsius for 10 minutes to denature to prepare a protein sample. . Then configure 10% PAGE gel (Yase, Shanghai, China) in the gel plate (Bole, CA, USA) according to the instructions, put the prepared gel in the electrophoresis tank (Bole, CA, USA), and connect the power supply (Bio-Rad, CA, USA) Let the bands run through the stacking gel at a constant voltage of 80 volts, and run through the separating gel at a constant voltage of 120 volts. When the strips reached the bottom of the separating gel, transfer the membrane using the wet method, and transfer the membrane at a constant current of 350 mA for 90 minutes in a transfer tank (Bio-Rad, CA, USA). After the membrane transfer was completed, the membrane was sheared according to the mass of Akt, ZAP70, ERK and GAPDH proteins. Block with rapid blocking solution (Yase, Shanghai, China) for ten minutes, and use pAkt-specific rabbit antibody (CST, MA, USA), Akt-specific rabbit antibody (CST, MA, USA), pZAP70-specific rabbit antibody (CST , MA, USA), ZAP70-specific map antibody (CST, MA, USA), pERK-specific rabbit antibody (CST, MA, USA), ERK-specific rabbit antibody (CST, MA, USA), GAPDH antibody (Conson, Shanghai, China) to incubate the corresponding bands separately, four times overnight. After washing with TBST the next day, incubate with specific anti-rabbit secondary antibody (Consun, Shanghai, China) diluted in 5% skimmed milk powder (Sangon, Shanghai, China) dissolved in TBS for one hour at room temperature, wash with TBST, and place Exposure to a gel imager (Bio-Rad, CA, USA) for one minute in a mixed luminescence solution (San Er, Shanghai, China).

Fig. 42 is the change of phosphorylation pathway shown by immunoblotting in Jurkat cells of CD3 mutants under the action of ITPRIPL1-RBD1-Fc protein. The results showed that the decreased phosphorylation caused by ITPRIPL1 in wild-type Jurkat cells was not observed in CD3 knockout/K76T mutant/ΔPRS mutant Jurkat cells, indicating that the regulation of ITPRIPL1 on the phosphorylation pathway of T cells requires a conformational change of CD3 with the PRS segment.

3. Intracellular calcium ion current fluorescent staining experiment showed that ITPRIPL1 can reduce the activity of T cells

The cultured Jurkat cells (ATCC, VA, U.S.) were counted, and ⁵ ×10 cells were transferred to each well of a 12-well plate (Corning, NY, U.S.), and treated with PBS or 2 micrograms per milliliter of ITPRIPL1-RBD protein for 24 hours. After the treatment, 4 micromolar Fluo-8AM (Abcam, MA, USA) intracellular calcium ion indicator was used for staining, and incubated in the incubator for 1 hour in the dark. HHBS buffer (Solarbio, Beijing, China) was washed twice, and observed under a fluorescent microscope.

Figure 43 is the signal intensity of intracellular calcium ion flow observed under a fluorescence microscope. The experimental results show that ITPRIPL1 has the function of significantly reducing the calcium ion flow in Jurkat cells, indicating that ITPRIPL1 has the function of reducing the activity of T cells.

4. Fluorescence staining experiments of intracellular calcium ion flow showed that ITPRIPL1 has the effect of reducing T cell activity, which depends on the conformational change of CD3 and the PRS segment

Count wild-type Jurkat cells, CD3 knockout Jurkat cells, CD3-K76T Jurkat cells, and CD3-ΔPRS cells, and take ^5x105 cells into each well of a 12-well plate (Corning, NY, USA), and give PBS or 4 micrograms Treatment was performed for 24 hours per ml of ITPRIPL1-RBD1-Fc protein. After the treatment, 4 micromolar Fluo-8AM (Abcam, MA, USA) intracellular calcium ion indicator was used for staining, and incubated in the incubator for 1 hour in the dark. HHBS buffer (Solarbio, Beijing, China) was washed twice, and observed under a fluorescent microscope.

Figure 44 shows the signal intensity of calcium ion flow in each cell observed under the fluorescence microscope. The experimental results showed that no significant effect of ITPRIPL1 on calcium ion flux in wild-type Jurkat cells was observed in CD3 knockout/K76T mutant/ΔPRS mutant Jurkat cells, indicating that ITPRIPL1's signal regulation on T cells requires conformational changes of CD3 and PRS part.

Example 16: ITPRIPL1 regulates T cell signaling and function by regulating CD3-Nck binding

1. Western blot experiments proved that ITPRIPL1-RBD1-Fc protein can increase the binding signal of CD3 and Nck in a concentration-dependent manner.

The cultured Jurkat cells (ATCC, VA, U.S.) were counted, and 5× ¹⁰ cells were transferred to a 15ml centrifuge tube (Corning, NY, U.S.), discarded after centrifugation at 800 rpm for 4 minutes, and replaced with serum-free RPMI1640 medium (U.S. Lun, Dalian, China) was resuspended, placed in an incubator for starvation culture for 3 hours, centrifuged at 800 rpm for 4 minutes, 5 ml of complete medium (Meilun, Dalian, China) was resuspended, and 1 ml each was placed in a 12-well plate (Corning, NY, USA) to each well, first add 10 micrograms per milliliter of OKT3 for activation treatment, and at the same time add: no treatment; 0.5/1/2/4 micrograms per milliliter of RBD1-Fc protein. After mixing thoroughly, place in the incubator and incubate for 5 minutes, take it out, transfer the cells to a 1.5ml EP tube (Axygen, CA, USA), centrifuge at 800rpm for 4 minutes, resuspend and wash with PBS, and repeat the washing. After the centrifugation was completed, the supernatant was discarded, and the immunoprecipitation lysate (Thermo Fisher, MA, USA) was mixed with protease inhibitor, phosphatase inhibitor, and PMSF (Consun, Shanghai, China) at a ratio of 1:100. µl of mixed lysate. Part of the cell samples were centrifuged, mixed with loading buffer (Beyond, Shanghai, China) and denatured in a metal bath at 100 degrees Celsius to obtain input protein samples; the remaining cell samples were treated with CD3E-specific mouse antibody (Santa cruz biotechnology, CA, USA) ) for immunoprecipitation, washed with PBS, mixed with loading buffer (Beyond, Shanghai, China) and denatured in a metal bath at 100 degrees Celsius to prepare immunoprecipitated protein samples. Supernatant after centrifugation: 5x loading buffer (Beyond, Shanghai, China) was configured at a ratio of 4:1 to each cell sample, and placed in a metal bath at 100 degrees Celsius for 10 minutes to denature to prepare a protein sample. Then configure 10% PAGE gel (Yase, Shanghai, China) in the gel plate (Bole, CA, USA) according to the instructions, put the prepared gel in the electrophoresis tank (Bole, CA, USA), and connect the power supply (Bole, CA, U.S.) Let the strips run through the stacking gel with a constant voltage of 80 volts, and run through the separating gel with a constant voltage of 120 volts. When the strips reached the bottom of the separating gel, transfer to the membrane using a wet method in a transfer tank (Bio-Rad, CA, USA) with a constant current of 350 mA for 90 minutes. After the membrane transfer was completed, the membrane was sheared according to the mass of CD3E and Nck proteins. Block with fast blocking solution (Yase, Shanghai, China) for ten minutes, and incubate corresponding bands with CD3E-specific rabbit antibody (CST, MA, USA) and Nck-specific rabbit antibody (CST, MA, USA) respectively, four degrees overnight. After washing with TBST the next day, incubate with specific anti-rabbit secondary antibody (Consun, Shanghai, China) diluted in 5% skimmed milk powder (Sangon, Shanghai, China) dissolved in TBS for one hour at room temperature, wash with TBST, and place Exposure to a gel imager (Bio-Rad, CA, USA) for one minute in a mixed luminescence solution (San Er, Shanghai, China).

As shown in Figure 45, the above results indicated that ITPRIPL1-RBD1-Fc protein can up-regulate the binding of CD3 and Nck in a concentration-dependent manner. It can be concluded from the above that ITPRIPL1 can increase the binding signal of CD3 and Nck in a concentration-dependent manner.

2. Proximity ligation experiment proved that ITPRIPL1-RBD1-Fc protein can increase the binding signal of CD3 and Nck.

The cultured Jurkat cells (ATCC, VA, U.S.) were counted, and 2× ¹⁰ cells were put into a 15ml centrifuge tube (Corning, NY, U.S.), discarded supernatant after centrifuging at 800 rpm for 4 minutes, and replaced with serum-free RPMI1640 medium (U.S. Lun, Dalian, China) was resuspended, placed in an incubator for starvation culture for 3 hours, centrifuged at 800 rpm for 4 minutes, 2 ml of complete medium (Meilun, Dalian, China) was resuspended, and 1 ml each was placed in a 12-well plate (Corning, NY, USA) to each well, first add 10 micrograms per milliliter of OKT3 for activation treatment, and at the same time add: no treatment; 4 micrograms per milliliter of RBD1-Fc protein to each well. After being placed in the incubator for 5 minutes, the Jurkat cells were taken out, and the reagents in the kit and the CD3E-specific mouse antibody (Santa cruz biotechnology, CA, USA) were used according to the instructions attached to the adjacent link experiment kit (Merck, NJ, USA). Correlation was performed with Nck-specific rabbit antibody (CST, MA, USA). After sealing, observe under a fluorescent microscope.

As shown in Figure 46, the proximity junction experiment showed that ITPRIPL1-RBD1-Fc protein can significantly increase the binding of CD3 and Nck, thereby regulating T cell signaling and function.

Example 17: ITPRIPL1 has the function of regulating immunity and T cell function in vivo, and promotes immune escape of tumors

1. Construction of humanized CD3ε mouse MC38 subcutaneous xenograft tumor in vivo model

The constructed full-length ITPRIPL1-Flag plasmid and the empty pcDNA3.1 plasmid were transfected into MC38 cells (Kerafast, MA, USA), and cultured in an incubator for 24-48 hours, then 200 micrograms per milliliter of Genetic Geneticin (G418) (Gibco, CA, USA) was used for screening, and after 10-14 days until all the cells in the empty pcDNA3.1 plasmid transfection group died, the MC38-ITPRIPL1 stable transfection cells were obtained.

Humanized CD3ε mice aged 6-8 weeks (Southern model, Shanghai, China) were randomly divided into groups according to body weight, with 6 mice in each group. Count the MC38 wild-type and MC38-ITPRIPL1 stable transfection cells, and resuspend them in PBS to a cell density of 1.5x10 ⁷ per milliliter. The mice were shaved, and 1.5× ^{10 6} wild-type or ITPRIPL1-overexpressed MC38 cells were inoculated subcutaneously in the right armpit, which was the construction of an in vivo model of humanized CD3ε mouse MC38 subcutaneously transplanted tumors.

2. Overexpression of ITPRIPL1 significantly increases tumor growth in mice

After the in vivo model of humanized CD3ε mouse MC38 subcutaneously transplanted tumor was constructed, the tumor size was measured with a vernier caliper on the fifth day after inoculation, and the long and short diameters of the tumor were measured each time with 1/2*A*a* The formula in a calculates the tumor volume, which is measured every three days and the tumor volume is recorded. On the 23rd day after inoculation, the mice were sacrificed uniformly, and after the tumors were peeled off, the tumor weights were weighed and counted.

As shown in Figure 47, both tumor volume and tumor weight showed that overexpression of ITPRIPL1 can significantly increase tumor growth, and has functions in vivo. Since ITPRIPL1 is widely expressed and upregulated in human tumors (see other examples in this application), the results of this example suggest that ITPRIPL1 plays a role in promoting immune escape in human tumors. Therefore, the inhibitor against ITPRIPL1 has the effect of inhibiting tumor growth, and the modulator of ITPRIPL1 has application value in the preparation of tumor therapeutic drugs.

3. Flow cytometry shows that overexpression of ITPRIPL1 inhibits T cell activity in mice

At least 1 ml of peripheral blood was obtained from the mouse by means of cardiac blood collection. Mouse PBMC cells were obtained by using a mouse peripheral blood PBMC isolation kit (Solarbio, Beijing, China) and operating according to the corresponding instructions of the kit.

Mouse PBMCs were placed in EP tubes (Axygen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), and the centrifugation and washing were repeated. Dilute mouse CD8-APC antibody (Biolegend, CA, U.S.), mouse CD69-APC antibody (Biolegend, CA, U.S.), mouse CD137-APC antibody (Biolegend, CA, U.S.) each EP with cell staining buffer at 1:20 Add 200 microliters of the mixture to the tube to resuspend, and incubate at room temperature for 30 minutes with slow shaking. After centrifugation at 400 rcf for 5 minutes, discard the supernatant, resuspend and wash the cells with 500 microliters of cell staining buffer; repeat the centrifugation and washing. Add 200 microliters of cell staining buffer to each, transfer to a flow tube (Falcon, NY, USA) and put it on a machine (Miltenyi, Cologne, Germany).

As shown in Figure 48, according to the mean fluorescence intensity, overexpression of ITPRIPL1 can significantly reduce the expression of CD8, CD25, and CD139. The experimental results indicated that overexpression of ITPRIPL1 could inhibit the activity of T cells in mice.

4. Immunohistochemical staining showed that overexpression of ITPRIPL1 reduced T cell infiltration in tumor

The MC38 tumor tissue was stripped and then sectioned and embedded in paraffin (Bioos, Wuhan, China). The obtained paraffin sections were subjected to dewaxing, hydration, antigen retrieval and other treatments, and then incubated with mouse CD8 antibody (CST, MA, USA) in a wet box overnight. After rewarming on the next day, secondary antibody incubation was carried out according to the reagent instructions. After DAB staining (Solarbio, Beijing, China) and hematoxylin staining (Solarbio, Beijing, China), air-dry against the alcohol concentration gradient and seal the slides. After the slides were sealed, they were observed under a fluorescent microscope using natural light and photographed.

As shown in Figure 49, overexpression of ITPRIPL1 can significantly reduce the positive rate of CD8 in tumor tissues. The experimental results indicated that ITPRIPL1 overexpression can reduce the infiltration of T cells in MC38 tumor tissue.

5. Construction of ITPRIPL1 heterozygous/homozygous knockout mouse model in vivo

Using CRISPR/Cas9 technology, using non-homologous recombination to repair the introduction of mutations, resulting in a frame shift of the Itpripl1 gene protein reading frame and loss of function. The brief process is as follows: Cas9 mRNA and gRNA were obtained by in vitro transcription; Cas9 mRNA and gRNA were microinjected into fertilized eggs of C57BL/6J mice to obtain F0 generation mice. The positive F0 generation mice identified by PCR amplification and sequencing were mated with C57BL/6J mice to obtain 6 positive F1 generation mice. The mice with heterozygous knockout and homozygous knockout of ITPRIPL1 were obtained.

6. Flow cytometry shows that knockdown of ITPRIPL1 enhances T cell activity

Blood was collected from the mouse model and PBMCs were isolated. Perform a cell count on the cell suspension. After cell counting, dilute with PBS solution to adjust the concentration of cells to be detected to 10 ⁶ cells/mL. Take 200ul of cell suspension and centrifuge at 1000rpm for 5min (4°C). Resuspend the cells in 100ul PBS; set up the detection tube as follows:

Negative control tube: 100ul cell suspension;

CD39 detection tube: add 2ul of PD1 antibody to 100ul cell suspension, and mix gently;

CD73 detection tube: add 2ul of PD-L1 antibody to 100ul cell suspension, and mix gently;

Antibodies were all (Thermo Fisher, MA, USA).

Incubate at 4°C in the dark for 30 minutes. After the incubation, the flow cytometer was used for detection.

As shown in Figure 50, the inhibitory surface molecules CD39 and CD73 were significantly reduced after ITPRIPL1 knockout, suggesting that ITPRIPL1 knockout can enhance the activity of T cells in vivo, and verified the negative regulatory effect of ITPRIPL1 itself on T cells.

7. ELISA detection showed that knockout of ITPRIPL1 increased the secretion of immune activation cytokines

Mouse PBMCs were operated and treated according to the ELISA kit instructions (Abnova, CA, USA) of cytokines granzyme A, granzyme B, IL-2, TNF-alpha, and IL-21, and corresponding experimental results were obtained.

As shown in Figure 51, knockout of ITPRIPL1 increases the secretion of cytokines such as granzyme A, granzyme B, IL-2, TNF-alpha, and IL-21, indicating that the secretion of immune activation factors in vivo increases after knockout of ITPRIPL1, suggesting that ITPRIPL1 own immunosuppressive effect.

8. Immunohistochemical staining showed that ITPRIPL1 knockout increased T cell infiltration in testicular tissue

The mouse testis tissue was stripped, stained and sectioned (Kilton, Shanghai, China), and the corresponding results were observed under a fluorescent microscope.

As shown in Figure 52, immunohistochemical staining showed that after ITPRIPL1 was knocked out, the positive rates of CD3, CD4, and CD8 in the testis increased, suggesting that T cell infiltration increased. Correspondingly, heterozygous and homozygous knockout mice had abnormal sperm morphology, abnormal motility patterns, and significantly reduced motility, consistent with the significant inflammatory infiltration that occurred in the testis, serving as strong supporting evidence for autoimmune disorders in testicular tissue. The above results indicate that ITPRIPL1 negatively regulates T cell activity under physiological conditions in the testis, and ITPRIPL plays a key role in maintaining the immune immunity state of the testis. Therefore, the expression of ITPRIPL1 may be used to predict the occurrence of testicular autoimmune diseases and autoimmune infertility, and the regulation of ITPRIPL1 activity can be used for the intervention and treatment of autoimmune infertility.

Example 18: ITPRIPL1 is used as a biomarker to indicate the presence of tumor cells in vivo, to predict the progression and stage of tumors, to distinguish the boundaries of tumors and normal tissues.

The present application innovatively reveals that ITPRIPL1 plays an important role in tumor immune escape, and tumors with elevated ITPRIPL1 expression are suitable for using ITPRIPL1 inhibitors to inhibit tumor growth. Therefore, it is very important to detect the expression of ITPRIPL1. This application demonstrates the method of detecting the expression of ITPRIPL1 using a specific antibody, and reveals that the high expression of ITPRIPL1 can be used to indicate the existence of tumor cells in vivo, and can accurately mark the border between tumor tissue and normal tissue. In addition, ITPRIPL1 was also significantly correlated with tumor progression stage. details as follows:

The procedure for generating polyclonal antibody to ITPRIPL1 was described previously. Further, the monoclonal antibody of ITPRIPL1 is prepared by hybridoma screening, isolation and culture. Using the antibody of ITPRIPL1 to perform immunohistochemical staining on a variety of tumor tissue chips (Shanghai Xinchao), scan the pictures and count the expression level and distribution of ITPRIPL1. As shown in Figure 53, the protein level of ITPRIPL1 was significantly increased in many types of common cancer types, and it was significantly stronger than that in para-cancerous tissues. Meanwhile, ITPRIPL1 was significantly correlated with tumor progression and stage. For example, the expression in advanced breast cancer (stage 3C, 4) is significantly higher than that in early stage (stage 1); the expression in advanced lung cancer (stage 4) is significantly higher than that in early stage (stage 1A); in colorectal cancer and thyroid cancer ITPRIPL1 has the above-mentioned characteristics that advanced cancer is higher than early cancer. The results show that the ITPRIPL1 biomarker can be used to indicate the presence of tumor cells in vivo and can predict tumor progression and stage.

The staining of ITPRIPL1 in tumor tissues showed a very prominent consistency. First, ITPRIPL1 was generally significantly up-regulated in the above-mentioned various tumors, and only a few tumors had exceptions. Secondly, the expression of ITPRIPL1 in tumor tissues has a strong uniformity, that is, the expression in different parts of the primary tumor and in different cells of the metastases is relatively consistent. This feature can be observed in Figure 56. ITPRIPL1 is significantly expressed in tumor cells that have metastasized to lymph nodes and can be used to distinguish cancer cells from normal lymph node tissues; ITPRIPL1 is also significantly highly expressed in tumor cells that have metastasized distantly, so it can be used to distinguish metastatic foci from normal tissues. The above-mentioned characteristics of ITPRIPL1 make it suitable for distinguishing tumor and normal cells and tissues, and accurately marking the boundaries between tumor and normal cells and tissues. This marker is suitable for analysis of tumor size and degree of invasion before and after treatment. Therefore, ITPRIPL1 expression is an important companion diagnostic marker for ITPRIPL1 modulator drugs. At the same time, the low heterogeneity makes ITPRIPL1 a very potential biomarker for tumor diagnosis.

Example 19: Mouse hybridoma antibody specifically binds to ITPRIPL1

1. Preparation of ITPRIPL1 mouse hybridoma antibody

(1) Human ITPRIPL1-Fc recombinant protein (as shown in SEQ ID NO: 21) was used as an immunogen to immunize C57BL/6 mice, and multiple immunizations were performed to enhance the effect:

1) For the first immunization, antigen 50 μg/monkey, plus Freund’s complete adjuvant subcutaneous injection in multiple points, with an interval of 3 weeks;

2) For the second immunization, the dosage route is the same as above, with Freund's incomplete adjuvant added, and the interval is 3 weeks;

3) For the third immunization, the dose is the same as above, without adjuvant, and the interval of intraperitoneal injection is 3 weeks;

4) Booster immunization, dose 50 μg, intraperitoneal injection. Blood was collected 3 days after the last injection to measure its potency.

(2) After detecting that the immune effect meets the requirements, blood is collected multiple times, polyclonal antibody is separated, and polyclonal antibody is purified. The specific experimental steps include:

1) Prepare protein G sepharose CL-4B affinity column. Prepare 10mL of protein G sepharose CL-4B filler, mix equal volumes of filler and TBS buffer solution in a vacuum bottle, and stir. Vacuum for 15 minutes to remove air bubbles in the filling. Slowly add protein G sepharose CL-4B filler into the glass column, use the pump to control the filling speed to 1mL/min-2mL/min, avoid column drying, and use 10 times the bed volume and pre-cooled TBS buffer solution to balance the column;

2) Preparation of polyclonal antibodies. Thaw the polyclonal antibody slowly in ice water or in a 4°C refrigerator to avoid protein aggregation. Add solid sodium azide to a concentration of 0.05%, centrifuge at 15,000×g for 5 minutes at 4°C, remove the clarified polyclonal antibody and filter through a filter to remove excess fat;

3) Affinity chromatography. Dilute the antibody with TBS buffer solution at a ratio of 1:5, and then filter through a filter. Put the polyclonal antibody onto the column at a rate of 0.5 mL per minute. In order to ensure the combination of the polyclonal antibody and the filler, it is necessary to load the column twice continuously and keep the sample effluent. Wash the column with TBS buffer solution until Aλ280nm<0.008, then add Ph 2.7 elution buffer solution, and elute at a speed of 0.5mL/min until all proteins flow down. Use a 1.5mL EP tube that has been added with 100 μL of neutralizing buffer solution to collect the eluate in separate tubes, and check the pH of the eluent with pH test paper after mixing. Antibody denaturation. Add 10 mL of elution buffer solution with pH 1.9 to the column, and collect the eluate according to the above method until Aλ280nm<0.008. The protein content in each tube was determined using a spectrophotometer.

2. Enzyme-linked immunosorbent assay (ELISA) verification of the binding of each hybridoma antibody to ITPRIPL1

In order to verify the binding ability of each hybridoma antibody to ITPRIPL1, enzyme-linked immunosorbent assay (ELISA) was used to confirm the direct binding of each antibody to ITPRIPL1 protein. A special plate for ELISA (costar, ME, USA) was used. First, 100 microliters (1 microgram per milliliter) of ITPRIPL1 recombinant protein (cusabio, Wuhan, China) ELISA coating solution (Solarbio, Beijing, China) was used to coat the plate, and the negative control was coated with 100 microliters of TPRIPL1 recombinant protein. Liquid cladding board. Four times wrapped overnight. After washing with PBST, 100 microliters of 5% BSA dissolved in PBS (VWR, PA, USA) was used to block, and the incubator was blocked at 37° C. for 90 minutes. After washing with PBST, bind in an incubator at 37°C for 60 minutes. After washing with PBST, specific anti-mouse Fc fragment antibody (Consun, Shanghai, China) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, prepare chromogenic solution (Invitrogen, CA, USA), 100 microliters per well, and place in an incubator for 5-30 minutes to react, then add 50 microliters of stop solution (Sangon, Shanghai, China), and place in Color reading at 450 nm on a microplate reader (Thermo Fisher, MA, USA).

Figure 57 shows the results of the ELISA experiment. The upper left figure shows the ELISA results of the binding of all 100 obtained antibodies to ITPRIPL1 protein, and the upper right figure shows the results of a separate repeated experiment of 9 antibodies with better binding selected from the results in the upper left figure. The figure below shows the concentration-dependent binding curve of 13B7 antibody. Experiments showed that the binding ability of each antibody to ITPRIPL1 was different, indicating the heterogeneity among antibodies, and 13B7 was the antibody with the strongest binding ability. The above results demonstrate that each hybridoma can bind to ITPRIPL1 with different binding abilities.

2. Flow cytometry (FACS) verification of the binding of each hybridoma antibody to ITPRIPL1

In order to further verify the binding ability of each hybridoma antibody to ITPRIPL1, flow cytometry (FACS) was used to confirm the direct binding of each antibody to ITPRIPL1 protein. Count the Jurkat cells (ATCC, VA, U.S.) that endogenously highly express ITPRIPL1, adjust the cell number to 1×10 ⁶ /ml, add 100 microliters to each 96-well plate (Thermo Fisher, MA, U.S.), and add to each The hybridoma antibody or control serum at a final concentration of 1 microgram per milliliter was added to the wells, and incubated in an incubator for 30 minutes. The 96-well plate was taken out, the cells in each well were placed in EP tubes (Axygen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), and the centrifugation and washing were repeated. Anti-mouse Fc fragment-Alexa Fluor 488 antibody (Invitrogen, CA, USA) was diluted 1:500 with cell staining buffer, 200 microliters of the mixture was added to each EP tube to resuspend, and incubated at room temperature for 30 minutes. After centrifugation at 400rcf for 5 minutes, discard the supernatant, resuspend and wash the cells with 1 ml of cell staining buffer; repeat the centrifugation and washing. Add 300 microliters of cell staining buffer to each, transfer to a flow tube (Falcon, NY, USA) and test on a machine (Miltenyi, Cologne, Germany).

Figure 58 is the result of the FACS experiment, which shows that the binding ability of each antibody to ITPRIPL1 is different, showing the heterogeneity among antibodies, among which 13B7 is the antibody with the strongest binding ability.

Example 20: 13B7 antibody can specifically bind ITPRIPL1 with high affinity

1. Flow cytometry (FACS) to verify the binding of 13B7 antibody to cells with different expression levels of ITPRIPL1

In order to further verify the binding ability of 13B7 to ITPRIPL1, flow cytometry (FACS) was used to confirm that the 13B7 antibody directly binds to various types of cells with different expression levels of ITPRIPL1. Cells with different expression levels of endogenous ITPRIPL1, namely HCT116 (ATCC, VA, USA), A549 (ATCC, VA, USA), MC38 (Kerafast, MA, USA), Jurkat (ATCC, VA, USA), Raji cells (ATCC, VA, U.S.) and MC38-ITPRIPL1 stable transfection strain cell count, adjust the cell number to be 1x10 ⁶ / milliliter, each add 100 microliters to the 96-well plate (Thermo Fisher, MA, U.S.), add to each well Add 13B7 antibody at a final concentration of 1 microgram per milliliter, and incubate in an incubator for 30 minutes. The 96-well plate was taken out, the cells in each well were placed in EP tubes (Axygen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), and the centrifugation and washing were repeated. Anti-mouse Fc fragment-Alexa Fluor 488 antibody (Invitrogen, CA, USA) was diluted 1:500 with cell staining buffer, 200 microliters of the mixture was added to each EP tube to resuspend, and incubated at room temperature for 30 minutes. After centrifugation at 400rcf for 5 minutes, discard the supernatant, resuspend and wash the cells with 1 ml of cell staining buffer; repeat the centrifugation and washing. Add 300 microliters of cell staining buffer to each, transfer to a flow tube (Falcon, NY, USA) and test on a machine (Miltenyi, Cologne, Germany).

Figure 59 is the result of the FACS experiment, which shows that the binding force between each cell line and the 13B7 antibody is basically consistent with the expression of ITPRIPL1 protein in each cell line, indicating that the 13B7 antibody can specifically bind to ITPRIPL1 on the cell line expressing ITPRIPL1 combined.

2. Flow cytometry (FACS) verified that 13B7 antibody can bind Jurkat cells in a concentration-dependent manner

To verify the binding ability of 13B7 to ITPRIPL1, flow cytometry (FACS) was used to verify the concentration-dependent binding of 13B7 antibody to Jurkat cells. Count the Jurkat cells endogenously highly expressing ITPRIPL1, adjust the cell number to 1×10 ⁶ /ml, add 100 microliters to a 96-well plate (Thermo Fisher, MA, USA), and add a final concentration of 0.0625/0.125/0.25/0.5/1/2 micrograms per milliliter of 13B7 antibody, incubated in the incubator for 30 minutes. The 96-well plate was taken out, the cells in each well were placed in EP tubes (Axygen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), and the centrifugation and washing were repeated. Anti-mouse Fc fragment-Alexa Fluor 488 antibody (Invitrogen, CA, USA) was diluted 1:500 with cell staining buffer, 200 microliters of the mixture was added to each EP tube to resuspend, and incubated at room temperature for 30 minutes. After centrifugation at 400rcf for 5 minutes, discard the supernatant, resuspend and wash the cells with 1 ml of cell staining buffer; repeat the centrifugation and washing. Add 300 microliters of cell staining buffer to each, transfer to a flow tube (Falcon, NY, USA) and test on a machine (Miltenyi, Cologne, Germany).

Figure 60 is the result of the FACS experiment, which shows that Jurkat cells can bind to the 13B7 antibody with high affinity in a concentration-dependent manner.

3. Western blot method to verify that the 13B7 antibody can specifically bind to the ITPRIPL1 protein of cells with different ITPRIPL1 expression levels

The Jurkat, HCT116, and MC38 cells containing endogenously expressed ITPRIPL1 were counted, and 1×10 ⁶ cells were taken out, washed with PBS and centrifuged, and then mixed with 60 microliters of RIPA lysate (Beiyuntian, Shanghai, China) and 1 : 100% protease inhibitor, phosphatase inhibitor, PMSF triple (Consun, Shanghai, China) mixed lysate lysed on ice for 15 minutes, took liquid nitrogen for three freezing and three thawing, after centrifugation, loading buffer (Biyuntian, Shanghai, China) to prepare input protein samples after mixing and denaturation in a metal bath at 100°C. Then configure 12.5% PAGE gel (Yase, Shanghai, China) in the gel plate (Bole, CA, USA) according to the instructions, place the prepared gel in the electrophoresis tank (Bole, CA, USA), and connect the power supply (Bole, CA, U.S.) Let the strips run through the stacking gel with a constant voltage of 80 volts, and run through the separating gel with a constant voltage of 120 volts. When the strips reached the bottom of the separating gel, transfer to the membrane using a wet method in a transfer tank (Bio-Rad, CA, USA) with a constant current of 350 mA for 90 minutes. After the membrane transfer was completed, the membrane was sheared according to the mass size of ITPRIPL1 protein and internal reference GAPDH protein. Block with fast blocking solution (Yase, Shanghai, China) for ten minutes, dilute with 13B7 antibody at a final concentration of 1 microgram per milliliter and incubate the corresponding bands of ITPRIPL1 and GAPDH with GAPDH-HRP antibody (Conson, Shanghai, China), 4°C overnight. After washing with TBST the next day, incubate with specific anti-mouse secondary antibody (Consun, Shanghai, China) diluted with 5% skimmed milk powder (Sangon, Shanghai, China) dissolved in TBS for one hour at room temperature, wash with TBST, and place Exposure to a gel imager (Bio-Rad, CA, USA) for one minute in a mixed luminescence solution (San Er, Shanghai, China).

Figure 61 is the result of the Western Blot experiment. As shown in the figure, according to the GAPDH internal reference, the 13B7 antibody can specifically display a band at the molecular weight position of the ITPRIPL1 protein, which is consistent with the endogenous expression of ITPRIPL1 in each cell, indicating that the 13B7 antibody can specifically bind to the ITPRIPL1 protein to combine.

Example 21: Determination of the blocking effect of each hybridoma antibody on the binding of ITPRIPL1 to CD3E/SEMA3G

In order to verify whether each hybridoma antibody has a blocking effect on the binding of ITPRIPL1 to CD3E or SEMA3G, an enzyme-linked immunosorbent assay (ELISA) was used to verify the blocking effect of each antibody. A special plate for ELISA (costar, ME, USA) was used. First, 1 microgram per milliliter of ITPRIPL1-Fc-tagged recombinant protein (Abclonal, Wuhan, China) or 1 microgram per milliliter of CD3E-Fc-labeled protein (Acro, Beijing, China) was dissolved in 100 microliters of ELISA coating solution (Solarbio, China). Beijing, China) and the negative control was coated with 100 microliters of coating solution without protein. Four times wrapped overnight. After washing with PBST, 100 microliters of 5% BSA dissolved in PBS (VWR, PA, USA) was used to block, and the incubator was blocked at 37 degrees for 90 minutes. After washing with PBST, SEMA3G-His tagged protein (cusabio, Wuhan, China) with a final concentration of 1 microgram per milliliter was added to the wells coated with ITPRIPL1-Fc protein, and a hybridoma antibody with a final concentration of 1/2 microgram per milliliter was added at the same time; Add ITPRIPL1-His tagged protein (cusabio, Wuhan, China) at a final concentration of 1 microgram per milliliter to the wells coated with CD3E-Fc protein, and at the same time add hybridoma antibody at a final concentration of 1/2 microgram per milliliter; incubator at 37 degrees Combine for 60 minutes. After washing with PBST, specific anti-His fragment antibody (Abcam, MA, USA) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, prepare chromogenic solution (Invitrogen, CA, USA), 100 microliters per well, and place in an incubator for 5-30 minutes to react, then add 50 microliters of stop solution (Sangon, Shanghai, China), and place in Color reading at 450 nm on a microplate reader (Thermo Fisher, MA, USA).

Figure 62 is the result of the ELISA experiment. The upper figure shows the blocking condition of each hybridoma antibody when ITPRIPL1-CD3E is combined, and the lower figure shows the blocking condition of each hybridoma antibody when ITPRIPL1-SEMA3G is combined. The experimental results showed that each hybridoma antibody could block the binding of ITPRIPL1 to CD3E or SEMA3G to varying degrees, showing the heterogeneity among the antibodies, among which the 18B12 and 13B7 antibodies had the best binding effects.

Example 22: Monoclonal antibodies can bind ITPRIPL1 with higher affinity

1. Immunosorbent assay (ELISA) to verify the binding ability of each monoclonal antibody to ITPRIPL1

After monoclonal purification of each hybridoma antibody, 16 monoclonal antibodies with better growth conditions were obtained. In order to verify the binding ability of each monoclonal antibody to ITPRIPL1, enzyme-linked immunosorbent assay (ELISA) was used to verify the direct binding of each antibody to ITPRIPL1 protein. A special plate for ELISA (costar, ME, USA) was used. First, 100 microliters of 1 microgram per milliliter ITPRIPL1 recombinant protein (cusabio, Wuhan, China) ELISA coating solution (Solarbio, Beijing, China) was used to coat the plate, and the negative control was coated with 100 microliters of protein-free coating solution. Four times wrapped overnight. After washing with PBST, 100 microliters of 5% BSA dissolved in PBS (VWR, PA, USA) was used to block, and the incubator was blocked at 37 degrees for 90 minutes. After washing with PBST, different monoclonal antibodies were added with a final concentration of 1 microgram per milliliter, and combined in an incubator at 37°C for 60 minutes. After washing with PBST, specific anti-mouse Fc fragment antibody (Consun, Shanghai, China) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, prepare chromogenic solution (Invitrogen, CA, USA), 100 microliters per well, and place in an incubator for 5-30 minutes to react, then add 50 microliters of stop solution (Sangon, Shanghai, China), and place in Color reading at 450 nm on a microplate reader (Thermo Fisher, MA, USA).

Figure 63 is the result of the ELISA experiment. The experimental results show that there is a certain gap in the binding ability of each monoclonal antibody to ITPRIPL1, and the overall binding ability to ITPRIPL1 is significantly stronger than that before monoclonal purification, indicating that the monoclonal antibody can bind ITPRIPL1 with a higher affinity.

2. Flow cytometry (FACS) to verify the binding of each monoclonal antibody to ITPRIPL1

In order to further verify the binding ability of each monoclonal antibody to ITPRIPL1, flow cytometry (FACS) was used to confirm the direct binding of each antibody to ITPRIPL1 protein. Count the Jurkat cells endogenously highly expressing ITPRIPL1, adjust the cell number to 1×10 ⁶ /ml, add 100 microliters to a 96-well plate (Thermo Fisher, MA, USA), and add a final concentration of 1 microgram per milliliter of monoclonal antibody or control serum, incubate for 30 minutes in the incubator. The 96-well plate was taken out, the cells in each well were placed in EP tubes (Axygen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), and the centrifugation and washing were repeated. Anti-mouse Fc fragment-Alexa Fluor 488 antibody (Invitrogen, CA, USA) was diluted 1:500 with cell staining buffer, 200 microliters of the mixture was added to each EP tube to resuspend, and incubated at room temperature for 30 minutes. After centrifugation at 400rcf for 5 minutes, discard the supernatant, resuspend and wash the cells with 1 ml of cell staining buffer; repeat the centrifugation and washing. Add 300 microliters of cell staining buffer to each, transfer to a flow tube (Falcon, NY, USA) and test on a machine (Miltenyi, Cologne, Germany).

The results are shown in Figure 64. The strength of the binding ability of each monoclonal antibody to ITPRIPL1 is different, showing the heterogeneity among antibodies, and the overall binding ability is stronger than that before monoclonal purification. The above results prove that each hybridoma can bind to ITPRIPL1 protein with different binding ability, and the binding ability to ITPRIPL1 is stronger after monoclonal purification.

Example 23: Determination of the blocking effect of each monoclonal antibody on the binding of ITPRIPL1 to CD3E/SEMA3G

In order to verify whether the obtained monoclonal antibody has a blocking effect on the binding of ITPRIPL1 to CD3E or SEMA3G, enzyme-linked immunosorbent assay (ELISA) was used to verify the blocking effect of each antibody. A special plate for ELISA (costar, ME, USA) was used. First, ITPRIPL1-Fc-tagged recombinant protein (Abclonal, Wuhan, China) at a final concentration of 1 μg/ml or CD3E-Fc-tagged protein (Acro, Beijing, China) at a final concentration of 1 μg/ml was dissolved in 100 μl ELISA The plate was coated with coating solution (Solarbio, Beijing, China), and the negative control was coated with 100 microliters of coating solution without protein. Coating overnight at 4°C. After washing with PBST, 100 microliters of 5% BSA dissolved in PBS (VWR, PA, USA) was used to block, and the incubator was blocked at 37 degrees for 90 minutes. After washing with PBST, SEMA3G-His tagged protein (cusabio, Wuhan, China) with a final concentration of 1 microgram per milliliter was added to the wells coated with ITPRIPL1-Fc protein, and a monoclonal antibody with a final concentration of 1 microgram per milliliter was added at the same time; CD3E-Fc protein wells were added with a final concentration of 1 microgram per milliliter of ITPRIPL1-His tagged protein (cusabio, Wuhan, China), while adding a final concentration of 1/2 microgram per milliliter of monoclonal antibody; minute. After washing with PBST, specific anti-His fragment antibody (Abcam, MA, USA) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, prepare chromogenic solution (Invitrogen, CA, USA), 100 microliters per well, and place in an incubator for 5-30 minutes to react, then add 50 microliters of stop solution (Sangon, Shanghai, China), and place in Color reading at 450 nm on a microplate reader (Thermo Fisher, MA, USA).

Figure 65 is the result of the ELISA experiment. The experimental results showed that each monoclonal antibody still has some differences in the blocking effect of ITPRIPL1 on CD3E/SEMA3G, and the blocking effect of 13B7A6H3/18B12D1A6/18B12D1F7 is better, which is consistent with the conclusion before monoclonal purification.

Example 24: Determination of the binding of hybridoma antibody and monoclonal antibody to polypeptide segment

Based on the protein sequence of the extracellular segment of ITPRIPL1, a total of 17 segments were designed with a 1/3 overlapping sequence (GenScript, Nanjing, China). Its sequence is shown in SEQ ID NO:42-58. The resulting polypeptide fragments were dissolved in DMSO at a final concentration of 400 micrograms per milliliter, enzyme-linked immunosorbent assay (ELISA) was used to verify the antibodies of each hybridoma, and 4 monoclonal antibodies were selected to verify the binding of the polypeptide fragments. A special plate for ELISA (costar, ME, USA) was used. Firstly, the peptide segment with a final concentration of 1 microgram per milliliter was coated with 100 microliters of ELISA coating solution (Solarbio, Beijing, China), and the negative control was coated with 100 microliters of coating solution without protein. Four times wrapped overnight. After washing with PBST, 100 microliters of 5% BSA dissolved in PBS (VWR, PA, USA) was used to block, and the incubator was blocked at 37 degrees for 90 minutes. After washing with PBST, different hybridoma antibodies or monoclonal antibodies with a final concentration of 1 microgram per milliliter were added, and combined in an incubator at 37 degrees for 60 minutes. After washing with PBST, specific anti-mouse Fc fragment antibody (Consun, Shanghai, China) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, prepare chromogenic solution (Invitrogen, CA, USA), 100 microliters per well, and place in an incubator for 5-30 minutes to react, then add 50 microliters of stop solution (Sangon, Shanghai, China), and place in Color reading at 450 nm on a microplate reader (Thermo Fisher, MA, USA).

Figure 66 is the result of the ELISA experiment. Reads that bind to a specific peptide fragment more than three times higher than background and other peptide fragments are considered to bind to a specific peptide fragment. The experimental results show that the monoclonal antibodies produced by 13B7/18B12 with better blocking effects all bind to P8, while the hybridoma antibodies and monoclonal antibodies with no or poor blocking effects have no specific binding peptides, indicating that P8 as the dominant binding site.

Example 25: Determination of the promotion effect of ITPRIPL1 monoclonal antibody on PBMC cells killing tumor cells

Flow cytometry proved that the monoclonal antibody that blocks the combination of ITPRIPL1 and CD3E/SEMA3G can promote the killing of tumor cells by PBMC.

The revived PBMC cells were activated with 1 ug/mL Anti-CD3/CD28 (Invitrogen, CA, USA) 24 hours in advance. Count the PBMC cells and Raji cells, adjust the cell number to 4x10 ⁶ /ml or 1x10 ⁶ /ml respectively, add 100 microliters to a 96-well plate (Thermo Fisher, MA, U.S.), and add two different concentrations of monoclonal antibody or control serum, mixed and placed in the incubator for co-incubation for 6 hours. The 96-well plate was taken out, the cells in each well were placed in EP tubes (Axygen, CA, USA), centrifuged at 400 rcf for 5 minutes, and the supernatant was discarded. The washed cells were resuspended with 500 microliters of cell staining buffer (Invitrogen, CA, USA), and the centrifugation and washing were repeated. After centrifugation at 400 rcf for 5 minutes, the supernatant was discarded, and the cells were resuspended and washed with 1 ml of binding buffer (Beyond, Shanghai, China); the centrifugation and washing were repeated. Set up each experiment and control group, add 100 microliters of binding buffer, 5 microliters of Annexin V-FITC (Beyond, Shanghai, China) and 10 microliters of PI (Beyond, Shanghai, China) according to the conditions, and incubate at room temperature for 15 minutes , and then add 400 microliters of binding buffer respectively, transfer to the flow tube (Falcon, NY, the United States) and test it on the machine (Miltenyi, Cologne, Germany), the results are shown in Figures 67 and 68, the results show that, with Compared with the control, among the antibodies, the monoclonal antibody 13B7A6H3/18B12D1A6, which has the effect of blocking the binding of ITPRIPL1 to CD3E/SEMA3G and can bind to P8 (SEQ ID NO: 49), significantly increased the killing of Raji cells by PBMC.

According to sequencing, the heavy chain sequence of the monoclonal antibody 13B7A6H3 is shown in SEQ ID NO:22, the light chain sequence is shown in SEQ ID NO:23, and the heavy chain variable region VH sequence is shown in SEQ ID NO:24, The light chain variable region VL is shown in SEQ ID NO:25, the sequence of the heavy chain complementarity determining region HCDR1 is shown in SEQ ID NO:26, the sequence of the heavy chain complementarity determining region HCDR2 is shown in SEQ ID NO:27, and the heavy chain complementarity determining region HCDR2 is shown in SEQ ID NO:27. The sequence of the chain complementarity determining region HCDR3 is shown in SEQ ID NO:28, the sequence of the light chain complementarity determining region LCDR1 is shown in SEQ ID NO:29, the sequence of the light chain complementarity determining region LCDR2 is KV, and the sequence of the light chain complementarity determining region LCDR3 The sequence of the monoclonal antibody 18B12D1A6 is shown in SEQ ID NO:31; the heavy chain sequence of the monoclonal antibody 18B12D1A6 is shown in SEQ ID NO:32, the light chain sequence is shown in SEQ ID NO:33, and the heavy chain variable region VH sequence is shown in SEQ ID Shown in NO:34, the light chain variable region VL is shown in SEQ ID NO:35, the sequence of the heavy chain complementarity determining region HCDR1 is shown in SEQ ID NO:36, and the sequence of the heavy chain complementarity determining region HCDR2 is shown in SEQ ID NO Shown in: 37, the sequence of the heavy chain complementarity determining region HCDR3 is shown in SEQ ID NO: 38, the sequence of the light chain complementarity determining region LCDR1 is shown in SEQ ID NO: 39, the sequence of the light chain complementarity determining region LCDR2 is KV, The sequence of the light chain complementarity determining region LCDR3 is shown in SEQ ID NO:41.

Analyzing the similarity of the above antibody sequences (Fig. 65C), it can be seen that the sequence of light chain CDR1 is xSLxNSKGNTH (x represents any amino acid) and the similarity with 13B7A6H3 light chain CDR1 is 81.8%, and the sequence of light chain CDR3 is SQSTHxPYT and is similar to 13B7A6H3 light chain CDR1 similarity is 87.5%. The other CDR sequences are identical. Therefore, it can be predicted that the similarity between light chain CDR1 and CDR3 and 13B7A6H3 is higher than 80%, and the antibody can still have the activity of binding ITPRIPL1 under the condition that other CDR sequences are identical to 13B7A6H3. If the overall similarity of all CDR sequences is compared, it can be known that the sequences with an overall similarity of 94% to the heavy chain CDR1, CDR2, and CDR3 of 13B7A6H3 and the light chain CDR1, CDR2, and CDR3 can have the activity of binding to ITPRIPL1.

Example 26: Determination of the effect of point mutations in polypeptide segments on the binding of monoclonal antibodies

Immunosorbent assay (ELISA) was used to verify the effect of point mutations in polypeptide segments on monoclonal antibodies.

The specific amino acid sequence of the polypeptide segment P8 obtained before was subjected to alanine (A) mutation one by one, and the original alanine site was not mutated, and the obtained sequence was shown in SEQ ID NO:59-71. The obtained polypeptide segment and the non-mutated polypeptide segment were dissolved in DMSO at a final concentration of 400 micrograms per milliliter, and enzyme-linked immunosorbent assay (ELISA) was used to verify the binding to the 13B7A6H3 monoclonal antibody. A special plate for ELISA (costar, ME, USA) was used. Firstly, the polypeptide segment with a final concentration of 1 microgram per milliliter was coated with 100 microliters of ELISA coating solution (Solarbio, Beijing, China), and the negative control was coated with 100 microliters of coating solution without protein. Four times wrapped overnight. After washing with PBST, 100 microliters of 5% BSA dissolved in PBS (VWR, PA, USA) was used to block, and the incubator was blocked at 37 degrees for 90 minutes. After washing with PBST, add 13B7A6H3 monoclonal antibody at a final concentration of 1 microgram per milliliter, and combine in an incubator at 37 degrees for 60 minutes. After washing with PBST, specific anti-mouse Fc fragment antibody (Consun, Shanghai, China) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, prepare chromogenic solution (Invitrogen, CA, USA), 100 microliters per well, and place in an incubator for 5-30 minutes to react, then add 50 microliters of stop solution (Sangon, Shanghai, China), and place in Color reading at 450 nm on a microplate reader (Thermo Fisher, MA, USA).

Figure 69 is the result of the ELISA experiment. The experimental results show that the 13B7A6H3 antibody no longer binds to the polypeptide segment after mutations at positions 3(L), 7(F), and 10(R) in P8, while mutations at other positions do not affect the binding, indicating that 3(L), 10(R) 7(F) and 10(R) are the key sites where 13B7A6H3 monoclonal antibody binds to ITPRIPL1 protein.

Example 27: Epitope mapping method competitive analysis ITPRIPL1 monoclonal antibody grouping

Grouping analysis of ITPRIPL1 monoclonal antibody by epitope mapping.

The obtained ITPRIPL1 monoclonal antibodies were analyzed by epitope mapping method to analyze the competitive relationship between the currently obtained ITPRIPL1 monoclonal antibodies and grouped. A special plate for ELISA (costar, ME, USA) was used. First, ITPRIPL1 protein with a final concentration of 0.5 micrograms per milliliter was used to coat the plate with 100 microliters of ELISA coating solution (Solarbio, Beijing, China), and the negative control was coated with 100 microliters of coating solution without protein. Four times wrapped overnight. After washing with PBST, 100 microliters of 5% BSA dissolved in PBS (VWR, PA, USA) was used to block, and the incubator was blocked at 37 degrees for 90 minutes. After washing with PBST, add 0.5 microgram per milliliter of each ITPRIPL1 monoclonal antibody, and at the same time cross-add each ITPRIPL1 monoclonal antibody with a final concentration of 0.5 microgram per milliliter, and combine in an incubator at 37 degrees for 60 minutes. After washing with PBST, specific anti-mouse Fc fragment antibody (Consun, Shanghai, China) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, prepare chromogenic solution (Invitrogen, CA, USA), 100 microliters per well, and place in an incubator for 5-30 minutes to react, then add 50 microliters of stop solution (Sangon, Shanghai, China), and place in Color reading at 450 nm on a microplate reader (Thermo Fisher, MA, USA).

Fig. 70 is the result of ELISA experiment of epitope mapping method. The experimental results show that there is a competitive relationship between 13B7 and 18B12, there is a competitive relationship between 13H10, 15C9, and 16E1, and there is no competitive relationship between other antibodies. It is suggested that the current ITPRIPL1 monoclonal antibodies can be divided into four groups: (1) 13B7, 18B12; (2) 13H10, 15C9, 16E1; (3) 13E8; (4) 18G5

Example 28: Construction of humanized ITPRIPL1 antibody and determination of binding sites

1. Construction of humanized ITPRIPL1 antibody

According to the results of previous antibody sequencing, the paired sequence of SEQ ID NO:73-82 corresponding to the 13B7A6H3 monoclonal antibody was optimized and recoded using humanized gene expression to obtain the corresponding humanized sequence, and the sequence was inserted into the human The Fc segment corresponds to the sequence vector to obtain the humanized ITPRIPL1 antibody sequence plasmid. The humanized ITPRIPL1 antibody sequence plasmid was transfected into HEK293 cells and cultured. After 72 hours, the supernatant was collected, mixed with the balanced protein A magnetic beads at room temperature for 2 hours, flow-through, equilibrated/washed, and eluted in the chromatography column, and the product obtained after neutralization was the humanized ITPRIPL1 antibody .

2. Binding activity and site determination of humanized ITPRIPL1 antibody

A special plate for ELISA (costar, ME, USA) was used. First, the ITPRIPL1 P8 polypeptide with a final concentration of 1 microgram per milliliter was used to coat the plate with 100 microliters of ELISA coating solution (Solarbio, Beijing, China). The negative control was coated with 100 microliters of coating solution without protein, and the positive control was coated with 100 microliters of ELISA coating solution. Human ITPRIPL1 P8 polypeptide coated plate. Four times wrapped overnight. After washing with PBST, 100 microliters of 5% BSA dissolved in PBS (VWR, PA, USA) was used to block, and the incubator was blocked at 37 degrees for 90 minutes. After washing with PBST, chimeric and humanized 13B7A6H3 monoclonal antibodies were added at a final concentration of 0.5 micrograms per milliliter, and combined in an incubator at 37 degrees for 60 minutes. After washing with PBST, specific anti-human Fc fragment antibody (Abcam, MA, USA) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, prepare chromogenic solution (Invitrogen, CA, USA), 100 microliters per well, and place in an incubator for 5-30 minutes to react, then add 50 microliters of stop solution (Sangon, Shanghai, China), and place in Color reading at 450 nm on a microplate reader (Thermo Fisher, MA, USA).

Fig. 74 ELISA experiment results of binding of humanized antibody to ITPRIPL1 P8 polypeptide fragment. The experimental results showed that the obtained humanized antibodies were active and could specifically bind to the site of the ITPRIPL1 polypeptide segment bound by 13B7A6H3, indicating that the antibody construction was successful.

Example 29: Binding assay of monoclonal ITPRIPL1 antibody to cynomolgus monkey ITPRIPL1 protein

Immunosorbent assay (ELISA) verified that the 13B7A6H3 monoclonal antibody could bind to the corresponding polypeptide segment of cynomolgus monkey ITPRIPL1.

According to the NCBI protein database, the sequence of the cynomolgus ITPRIPL1 protein is known as SEQ ID NO: 72; wherein the sequence of the human ITPRIPL1 P8 corresponding to the cynomolgus monkey is SEQ ID NO:. The corresponding polypeptide is constructed according to the corresponding polypeptide segment of cynomolgus monkey. A special plate for ELISA (costar, ME, USA) was used. First, the cynomolgus monkey ITPRIPL1 polypeptide with a final concentration of 1 microgram per milliliter was used to coat the plate with 100 microliters of ELISA coating solution (Solarbio, Beijing, China), and the negative control was coated with 100 microliters of coating solution without protein. As a control, human ITPRIPL1 P8 polypeptide was used to coat the plate. Four times wrapped overnight. After washing with PBST, 100 microliters of 5% BSA dissolved in PBS (VWR, PA, USA) was used to block, and the incubator was blocked at 37 degrees for 90 minutes. After washing with PBST, 13B7A6H3 monoclonal antibody with a final concentration of 0.5 microgram per milliliter was added, and combined in an incubator at 37 degrees for 60 minutes. After washing with PBST, specific anti-mouse Fc fragment antibody (Consun, Shanghai, China) diluted in PBS was used for incubation and binding, and the incubator was combined at 37 degrees for 30 minutes. After washing with PBST, prepare chromogenic solution (Invitrogen, CA, USA), 100 microliters per well, and place in an incubator for 5-30 minutes to react, then add 50 microliters of stop solution (Sangon, Shanghai, China), and place in Color reading at 450 nm on a microplate reader (Thermo Fisher, MA, USA).

Figure 75 shows the results of ELISA experiments on the binding of cynomolgus monkey ITPRIPL1 polypeptide and ITPRIPL1 monoclonal antibody. The experimental results showed that there was no significant difference in the binding of cynomolgus monkey ITPRIPL1 polypeptide and human ITPRIPL1 P8 polypeptide to 13B7A6H3 monoclonal antibody, suggesting that 13B7A6H3 monoclonal antibody could bind to cynomolgus monkey ITPRIPL1 protein. .

In summary, the present application discloses a newly identified natural membrane protein ITPRIPL1 that binds to the extracellular region of CD3ε, and reveals the changes in signal transduction that control T cell activation after ITPRIPL1 binds to CD3ε. This suggests that the CD3ε-binding antibodies discovered in the past may mimic or affect the natural ligand function of ITPRIPL1 to a certain extent. The discovery of CD3ε and ITPRIPL1, a pair of immune checkpoint receptor ligands, brings a new perspective to the in-depth understanding of the maintenance of immune homeostasis and the immune escape mechanism of tumor cells.

Although the content of the present application has been described in detail through the above preferred embodiments, it should be understood that the above description should not be considered as limiting the present application. Various modifications and alterations to the present application will become apparent to those skilled in the art after reading the above disclosure. Therefore, the protection scope of the present application should be defined by the appended claims.

Claims (47)

1. Use of a modulator of ITPRIPL1 in the manufacture of a medicament for modulating an immune response or an anti-tumour, wherein the modulator is used to increase or decrease the expression or function of an ITPRIPL1 gene or protein in the body.
2. Use of a modulator of ITPRIPL1 according to claim 1 in the manufacture of a medicament for modulating an immune response or an anti-tumour, wherein the modulator comprises any one of:

   (1) A gene editing system that causes the intracellular ITPRIPL1 gene to be knocked out or mutated;

   (2) An RNA molecule that reduces the expression level of the ITPRIPL1 gene;

   (3) A nucleic acid molecule for introducing into a cell, said nucleic acid molecule encoding ITPRIPL1 and increasing the expression level of ITPRIPL 1;

   (4) An isolated ITPRIPL1 recombinant protein;

   (5) An antibody that recognizes and binds to ITPRIPL 1.
3. Use of a modulator of ITPRIPL1 according to claim 2 in the manufacture of a medicament for modulating an immune response or anti-tumour, wherein:

   the gene editing system is a CRISPR/Cas9 gene editing system; the target sequence for the CRISPR/Cas9 gene editing system is selected from any one sequence shown in SEQ ID NO. 11-13, and the oligo DNA sequence for encoding sgRNA is selected from SEQ ID NO. 14-19;

   the nucleic acid molecule comprises: SEQ ID NO. 8, SEQ ID NO. 9 or SEQ ID NO. 10;

   The ITPRIPL1 recombinant protein comprises: a functional fragment in the extracellular domain of the ITPRIPL1 protein capable of binding to CD3 epsilon or NRP2 protein;

   the antibody which recognizes and binds to the ITPRIPL1 is a polyclonal antibody, a monoclonal antibody, a single chain antibody, an antigen binding domain, a bispecific antibody, a multispecific antibody, or an antigen binding portion in a chimeric antigen receptor.
4. Use of a modulator of ITPRIPL1 according to claim 3 in the manufacture of a medicament for modulating an immune response or an anti-tumour, wherein the sequence of the functional fragment is selected from any one of SEQ ID No. 1 to SEQ ID No. 4, or is a derivative thereof comprising drmdldtlarqlekrmsemrexlemefexxxaexxqkexenxwxgxtsxdq ("x" is any amino acid), the method of derivatization comprising: 1-10 amino acids are replaced, deleted or inserted without changing the sequence function; the derivative sequence is preferably drmdldtlarsrqlekrmsemrsexlemefexxxaexxaexxqkxenxwxgxtsxdq ("x" is any amino acid).
5. Use of a modulator of ITPRIPL1 according to claim 3 or 4 in the manufacture of a medicament for modulating an immune response or an anti-tumour, wherein the recombinant protein of ITPRIPL1 forms a fusion protein with an antibody constant region or a fusion protein with a clotting factor; alternatively, the ITPRIPL1 recombinant protein is modified in a manner comprising: polyethylene glycol modification, glycosylation modification, polysialic acid modification, fatty acid modification, KLH modification and biotin modification.
6. Use of a modulator of ITPRIPL1 according to claim 3 in the manufacture of a medicament for modulating an immune response or an anti-tumour, wherein the nucleic acid molecule is introduced into a cell by a drug delivery system comprising: recombinant expression vectors, viruses, liposomes or nanomaterials.
7. Use of a modulator of ITPRIPL1 according to claim 1 in the manufacture of a medicament for modulating an immune response or an anti-tumour, wherein said modulating an immune response comprises: modulation of antigen presenting cell and T lymphocyte function during autoimmune reaction, inhibition of graft rejection immune reaction, allergic reaction, anti-infective immune reaction, anti-tumor immune reaction.
8. Use of a modulator of ITPRIPL1 according to claim 7 in the manufacture of a medicament for modulating an immune response or an anti-tumour, wherein the immune response comprises: type I diabetes, immune infertility, rejection after organ transplantation, allergic reactions, systemic inflammation or cytokine storm, infection.
9. Use of a modulator of ITPRIPL1 according to claim 1 in the manufacture of a medicament for modulating an immune response or for combating a tumour, wherein the tumour is an ITPRIPL 1-associated solid tumour or a hematological tumour; the solid tumor comprises: glioma, lung cancer, head and neck cancer, gastric cancer, colorectal cancer, thyroid cancer, esophageal cancer, urothelial cancer, testicular cancer, breast cancer, cervical cancer, endometrial cancer, melanoma, pancreatic cancer, or liver cancer; the hematological tumor comprises: leukemia or lymphoma.
10. A pharmaceutical composition comprising a modulator for use according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier.
11. An isolated recombinant ITPRIPL1 protein, wherein the recombinant protein is a functional fragment of the extracellular domain of the ITPRIPL1 protein that is capable of binding to CD3 epsilon or NRP2 protein.
12. Use of an antibody that recognizes and binds to ITPRIPL1 in the manufacture of a medicament for modulating an immune response or an anti-tumor, wherein said antibody recognizes and binds to an extracellular region of an ITPRIPL1 protein.
13. The use of antibodies that specifically recognize ITPRIPL1 to label the boundary of cancer tissue and paracancestral tissue within a primary focus, the boundary of cancer cells that metastasize to lymph nodes and normal lymphoid tissue, the boundary of cancer cells that metastasize distally and normal tissue of a metastatic organ, and to label viable cells of cancer tissue in other biological samples.
14. The use of claim 13, wherein the cancer comprises thyroid cancer, breast cancer, colorectal cancer, lung cancer, esophageal cancer, renal cancer, and other ITPRIPL 1-related tumors.
15. An isolated antigen ITPRIPL1 binding protein capable of binding to the amino acid sequence set forth in SEQ ID No. 49 or SEQ ID No. 1 in antigen ITPRIPL 1.
16. The isolated antigen ITPRIPL1 binding protein of claim 15, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region VH set forth in any one of amino acid sequences SEQ ID No. 24 or SEQ ID No. 34; the light chain variable region comprises the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region VL as set forth in either SEQ ID NO 25 or SEQ ID NO 35.
17. The isolated antigen ITPRIPL1 binding protein of claim 16, wherein the amino acid sequence of HCDR1 is shown in SEQ ID No. 26, the amino acid sequence of HCDR2 is shown in SEQ ID No. 27, and the amino acid sequence of HCDR3 is shown in SEQ ID No. 28.
18. The isolated antigen ITPRIPL1 binding protein of claim 16, wherein the amino acid sequence of HCDR1 is shown in SEQ ID No. 36, the amino acid sequence of HCDR2 is shown in SEQ ID No. 37, and the amino acid sequence of HCDR3 is shown in SEQ ID No. 38.
19. The isolated antigen ITPRIPL1 binding protein of claim 16, wherein the amino acid sequence of LCDR1 is more than 80% similar to the amino acid sequence of SEQ ID No. 29, the amino acid sequence of LCDR2 is KV, and the amino acid sequence of LCDR3 is more than 80% similar to the amino acid sequence of SEQ ID No. 31.
20. The isolated antigen ITPRIPL1 binding protein of claim 16, wherein the amino acid sequence of LCDR1 is shown as any one of SEQ ID No. 29 or SEQ ID No. 39, the amino acid sequence of LCDR2 is KV, and the amino acid sequence of LCDR3 is shown as any one of SEQ ID No. 31 or SEQ ID No. 41.
21. The isolated antigen ITPRIPL1 binding protein of claim 16, wherein the amino acid sequence of LCDR1 is shown in SEQ ID No. 29, the amino acid sequence of LCDR2 is KV, and the amino acid sequence of LCDR3 is shown in SEQ ID No. 31; or the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 39, the amino acid sequence of the LCDR2 is KV, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 41.
22. The isolated antigen ITPRIPL1 binding protein of claim 16, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No. 24; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID NO. 25 or SEQ ID NO. 35.
23. The isolated antigen ITPRIPL1 binding protein of any one of claims 15-22, comprising an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.
24. The isolated antigen ITPRIPL1 binding protein of any one of claims 15-22, comprising an antibody light chain constant region, and the antibody light chain constant region comprises a human igκ constant region.
25. The isolated antigen ITPRIPL1 binding protein of any one of claims 15-22, comprising an antibody heavy chain HC, and the HC comprises an amino acid sequence set forth in any one of SEQ ID NOs 22 or 32.
26. The isolated antigen ITPRIPL1 binding protein of any one of claims 15-22, comprising an antibody light chain LC and said LC comprising the amino acid sequence set forth in any one of SEQ ID NOs 23 or 33.
27. The isolated antigen ITPRIPL1 binding protein of any one of claims 15-22, comprising an antibody or antigen binding fragment thereof, wherein the antigen binding fragment comprises Fab, fab ', F (ab) 2, fv fragment, F (ab') 2, scFv, and/or di-scFv.
28. The isolated antigen ITPRIPL1 binding protein of any one of claims 15-22, the ITPRIPL1 protein comprising human and cynomolgus ITPRIPL1 proteins.
29. A chimeric antigen receptor comprising the isolated antigen ITPRIPL1 binding protein of any one of claims 15-28.
30. An immunoconjugate comprising the isolated antigen ITPRIPL1 binding protein of any one of claims 15-28.
31. An isolated one or more nucleic acid molecules encoding the isolated antigen ITPRIPL1 binding protein of any one of claims 15-28 or the chimeric antigen receptor of claim 29, or encoding the polypeptide or protein fragment of any one of claims 4, 11.
32. A vector comprising the nucleic acid molecule of claim 31.
33. A cell comprising the nucleic acid molecule of claim 31 or the vector of claim 32.
34. A pharmaceutical composition comprising the isolated antigen ITPRIPL1 binding protein of any one of claims 15-28, the chimeric antigen receptor of claim 29, the immunoconjugate of claim 30, and optionally a pharmaceutically acceptable adjuvant.
35. Culturing the cell of claim 33 under conditions in which the isolated antigen ITPRIPL1 binding protein of any one of claims 15-28 is expressed.
36. Use of the isolated antigen ITPRIPL1 binding protein of any one of claims 15-28, the chimeric antigen receptor of claim 29, the immunoconjugate of claim 30, and/or the pharmaceutical composition of claim 34 in the manufacture of a medicament for preventing, alleviating and/or treating a tumor.
37. The use of claim 36, wherein the tumor comprises an ITPRIPL 1-related tumor.
38. A method of screening and identifying ITPRIPL 1-associated tumors by detecting expression of ITPRIPL1 in a biological sample taken from a subject, said detection comprising detection by antibodies, nucleic acid probes or PCR reactions comprising specific primers, and the like, and comparing said expression with a predetermined value, said biological sample comprising samples of tissue, blood, urine, cerebrospinal fluid, ascites, hydrothorax, tissue exudates, stool, and the like.
39. The method of claim 38, use in the manufacture of a kit for tumor screening or diagnosis, and use in the manufacture of a companion diagnostic kit for screening patients for an ITPRIPL1 modulator.
40. A linear epitope polypeptide of ITPRIPL1, the sequence of the linear epitope peptide comprising:

    the amino acid sequence of (i) is SEQ ID NO. 49, namely RLLEMEFEERKRAAE;

    (ii) either the amino acid sequence is or xxLxxxFxxRxxx (x is any amino acid), 1 to 3 amino acids at both ends may be deleted;

    (iii) Or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of SEQ ID NO. 49;
41. A nucleic acid molecule comprising a nucleic acid sequence encoding a peptide according to claim 40 or a complement thereof.
42. A vector comprising the nucleic acid molecule of claim 41.
43. A host cell comprising the vector of claim 42.
44. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a peptide according to claim 40, a carrier according to claim 42 or a cell according to claim 43.
45. The epitope peptide of claims 40-44, and corresponding encoding nucleic acids, vectors, host cells, pharmaceutical compositions, and uses thereof in screening and preparing ITPRIPL1 modulators.
46. A method for the discovery and identification of modulators of the ITPRIPL1 function according to claims 1-36, characterized in that one or more of the following properties of the test molecule are detected: the ability to specifically bind cell surface expressed ITPRIPL 1; effects on binding of ITPRIPL1 to CD3 epsilon; effects on binding of ITPRIPL1 to NRP 2; effects on binding of ITPRIPL1 to SEMA 3G; effects on binding of ITPRIPL1 to EBI 2; the effect on immune or tumor cell function, either directly or indirectly, may be a detectable effect in vitro or in vivo.
47. Use of an isolated antigen ITPRIPL1 binding protein capable of binding to an amino acid sequence shown in SEQ ID No. 49 or SEQ ID No. 1 in antigen ITPRIPL1 in the preparation of a medicament for modulating an immune response or an anti-tumour and detecting expression of ITPRIPL1 in a subject.